Method for identifying modulators of G protein coupled receptor signaling

ABSTRACT

This invention relates to methods for identifying peptides and other compounds which block or enhance G protein coupled receptor mediated signaling with high affinity and specificity and/or which stabilize a particular conformer of a G protein coupled receptor. Assays, methods of treatment and other methods developed in conjunction with these methods also are disclosed.

This application is a continuation of application Ser. No. 10/411,336,filed Apr. 11, 2003, which is a continuation-in-part of prior co-pendingapplication Ser. No. 09/852,910, filed May 11, 2001, which claimspriority from prior co-pending provisional application Ser. No.60/275,472, filed Mar. 14, 2001.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention generally pertains to the field of modulatingactivity of G protein-coupled receptors (GPCR) and of identifying andpreparing G protein coupled receptor antagonist and agonist compounds,including direct, indirect, full, partial, inverse and allostericagonists. The invention also encompasses compounds that bind to GPCR tostabilize a particular conformation of the GPCR. These compounds canserve as lead compounds for drug discovery purposes or for studying theGPCR three dimensional structure of specific conformations by suchmethods as X-ray crystallography or NMR. The invention also relates toan approach using high-throughput screening to identify small moleculesthat can bind to GPCRs and modulate their function by affecting the wayin which they contact their cognate G protein(s). As a first step inidentifying GPCR modulators, peptide analogs are identified that mimicor antagonize G proteins and bind with high affinity to the particularreceptor under study. These peptides then are tested for theirspecificity. The most specific peptides are used in a competitive assayto screen for small molecules or other peptides that can, for example,(1) increase the binding of the high affinity peptide (“super agonist”)or (2) can decrease the binding of the high affinity peptide, presumablyby competing for binding at the GPCR (“antagonists”).

2. Description of the Background Art

A great number of chemical messengers exert their effects on cells bybinding to G protein-coupled receptors (GPCR). GPCR include a wide rangeof biologically active receptors such as hormone receptors, viralreceptors, growth factor receptors, chemokine receptors, sensorreceptors and neuroreceptors. These receptors are activated by thebinding of ligand to an extracellular binding site on the GPCR andmediate their actions through the various G proteins. The molecularinteractions that occur between the receptor and the G protein arefundamental to the transduction of environmental signals into specificcellular responses.

G protein-coupled receptors have seven transmembrane helices which form,on the intracellular side of the membrane, the G protein binding domain.Experiments have suggested that activation of the receptor by ligandbinding changes conformation of the receptor, unmasking G proteinbinding sites on the intracellular face of the receptor. Thetransduction of the signal from the extracellular to intracellularenvironments requires the actions of heterotrimeric G proteins. Themolecular interactions that occur between the receptor and the G proteinare fundamental to the transduction of environmental signals intospecific cellular responses. Heterotrimeric G proteins are thought tointeract with GPCR in a multi-site fashion with the major site ofcontact being at the carboxyl terminus of the Gα subunit. Hamm et al.,Science 241:832-835, 1998; Osawa and Weiss, J. Biol. Chem.270:31052-31058, 1995; Garcia et al., EMBO J. 14:4460-4469, 1995;Sullivan et al., J. Biol. Chem. 269:21519-21525, 1994; West et al., J.Biol. Chem. 260:14428-14430, 1985.

In the inactive state, G proteins are heterotrimeric, consisting of oneα, one β and one γ subunit and a bound deoxyguanosine diphosphate (GDP).Following ligand binding, the GPCR becomes activated. Conformationalchanges in the activated receptor lead to activation of the G protein,with subsequent decreased affinity of Gα for GDP, dissociation of theGDP and replacement with GTP. Once GTP is bound, Gα assumes its activeconformation, dissociates from the receptor, and activates a downstreameffector. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself,returns the G-protein to its basal, inactive form. Thus, the G-proteinserves a dual role, as both an intermediate that relays the signal fromreceptor to effector and as a clock that controls the duration of thesignal. A variety of studies have implicated the carboxyl terminus of Gprotein α subunits in mediating receptor-G protein interaction andselectivity.

The carboxyl terminal 11 amino acids are most important to receptorinteraction and to the specificity of this interaction. Martin et al.,J. Biol. Chem. 271:361-366, 1996; Kostenis et al., Biochemistry36:1487-1495, 1997. Other regions on Gα also are involved in receptorcontact, however. Portions of the Gβγ dimer also have been implicated inGPCR binding. See Onrust et al., Science 275:381-384, 1997; Lichtarge etal., Proc. Natl. Acad. Sci. USA 93:7507-7611, 1996; Mazzoni and Hamm, J.Biol. Chem. 271:30034-30040, 1996; Bae et al., J. Biol. Chem.272:32071-32077, 1997. The carboxyl terminal amino acid regions of Gαproteins (and other GPCR binding regions of the heterotrimeric Gprotein) not only provide the molecular basis of receptor-mediatedactivation of G proteins, but also play an important role in determiningthe fidelity of receptor activation. Conklin et al., Nature 363:274-276,1993; Conklin et al., Mol. Pharmacol. 50:885-890, 1996.

The involvement of the carboxyl-terminal 11 amino acids of Gt (aminoacids 340-350) in interactions with the activated GPCR (R*) is suggestedby many studies, including (a) the finding that Pertussis toxincatalyzes the ADP-ribosylation of Cys0347, which uncouples Gt from R*;(b) a peptide corresponding to amino acids 340-350 of Gt can uncouple R*from Gt and can itself bind to R* and mimic the effects of Gt; (c)site-directed mutagenesis; and (d) the demonstration in related Gproteins that specificity of coupling to particular receptors resides intheir carboxyl terminus in interacting with R*.

The G proteins play important and intricate roles in determining thespecificity and temporal characteristics of the cellular response to theligand-binding signal. Hamm and Gilchrist, Curr. Opin. Cell Biol.8:189-196, 1996. Multiple receptors can activate a single G proteinsubtype, and in some cases a single receptor can activate more than oneG protein, thereby mediating multiple intracellular signals. In othercases, however, interaction of a receptor with a G protein is regulatedin a highly selective manner such that only a particular heterotrimer isbound.

Recognition sites are the precise molecular regions on receptors towhich the activating molecules bind. An agonist is an endogenoussubstance or a drug that can interact with a receptor and initiate aphysiological response. A drug may interact at the same site as anendogenous agonist (i.e., hormone or neurotransmitter) or at a differentsite. Agonists that bind to an adjacent or a different site are termedallosteric agonists. As a consequence of the binding to allostericbinding sites, the interaction with the normal ligand may be eitherenhanced or reduced. The conformational change which the allostericmodulators induce in receptors concerns not only the binding domain forthe classical ligands, but also the domain responsible for theinteraction between the receptors and the G proteins.

The visual system is an example of one in which G protein signaling isimportant. Rod cells of the retina make up 95% of the photoreceptors andare highly sensitive to light. Rods allow vision at night or underconditions of very dim illumination. The rod visual protein rhodopsinresides in disk membranes in the rod outer segment (ROS). Rhodopsin is aprototypical GPCR. Helmreich and Hofmann, Biochim. Biophys. Acta1286:285-322, 1996; Menon et al., Physiol. Rev. 81:1659, 2001; Teller etal., Biochemistry 40:7761, 2001. Rhodopsin is unique among GPCRs as itis not ligand activated.

Night vision relates to the ability of the organism to discriminatebetween slight differences in the intensity of dim light and, whendark-adapted, to detect small changes in light. Some persons reportconsistent difficulties in seeing at night, even when their eyes arefully dark-adapted. They cannot detect objects readily visible to othersand show both confusion and slow recovery after brief exposure torelatively bright light sources. Maneuvering in dimly illuminated spacesand driving or flying at night present serious problems to theseindividuals. In addition, some individuals have nyctalopia, or truenight blindness, which is diagnosed on the basis of a measurement ofretinal sensitivity.

No definitive data on the occurrence of nyctalopia in the population areavailable, since measurements have never been made on a representativesample of the population. Studies of select groups (e.g., schoolchildren, service men), show that the normal population includes apercentage of persons of low visual sensitivity whose performance willbe as poor as or poorer than that of many individuals whose nyctalopiais associated with disease or degenerative processes. For example, about2 percent of Navy men were disqualified for night duties as “nightblind” on this basis. It is also a disease of aging. As the generalpopulation ages, incidence of night blindness increases. Night blindnessalso has been observed in several diseases including: (1) Retinitispigmentosa (In the early stages of the disease, dark adaptation takesplace, but at a retarded rate. As disease advances, rod function isprogressively lost, and the absolute terminal threshold is elevated.More than 100,000 Americans have retinitis pigmentosa, and most peoplewith retinitis pigmentosa are blind by the age of 40. See Farrar et al.,EMBO J. 21(5):857-864, 2002; (2) Glaucoma (Early impairment andprogressive loss of rod sensitivity is observed in glaucoma. Cursiefenet al., Doc. Ophthalmol. 103(l):1-12, 2001. Glaucoma is one of theleading causes of blindness in the U.S and one of the most common causesof blindness in individuals over age 60, one of the fastest growinggroups in the U.S.); (3) LASIK (Recent studies indicate a significantnumber of patients who undergo LASIK surgery fail a night vision test(30-60%). Miller et al., CLAO J. 27:84-88, 2001; Brunette et al.,Ophthalmology 107:1790-1796, 2000; (4) Side effects of drugs (Severalmedications can cause night blindness, including Methyltestosterone,Quinidinesis, Paramethadion and Trimethadione (anticonvulsants),Questran (cholesterol-lowering), Accutane (anti-acne),Hydroxychloroquine (anti-malarial), Videx (HIV), and Nefazodone(antidepressant)). Thus, the usefulness of a pharmaceutical approach tonight blindness is clear. As the population ages, the number of affectedindividuals will increase.

Human dietary vitamin A deficiency can cause night blindness, and thiscan be reversed with vitamin A supplements. However, the night blindnessassociated with visual diseases such as retinitis pigmentosa (RP),cataracts, diabetic retinopathy, and glaucoma is only somewhat helpedwith vitamin A supplements, which do not change the course of thedisease. Many of the mutations that cause retinal degeneration andvisual loss are in genes that encode photoreceptor cascade proteins;others are in genes that encode photoreceptor structural proteins. Pangand Lam, Hum. Mutat. 19:189, 2002. Mutations in rhodopsin, PDEβ, or Gαthave been identified in different forms of congenital stationary nightblindness. Pepe, Prog. Retin. Eye Res. 20:733-759, 2001. Stationarynight blindness is not associated with retinal degeneration andmanifests itself in the inability to see in the dark; daytime vision islargely unaffected. Congenital stationary night blindness (CSNB) refersto a group of non-progressive retinal disorders that are characterizedpredominantly by abnormal function of the rod system. Clinicalheterogeneity even among family members with the same mutation raisesthe possibility that modifying factors, either genetic or environmental,influence the severity of the disease. Gottlob, Curr. Opin. Ophthalmol.12:378-383, 2001.

In night blindness resulting from defects in rhodopsin, Gαt, or PDEβ,rod photoreceptors respond only to light intensities far brighter thannormal, and the sensitivity of rods to light is similar to that ofnormal individuals who are not dark adapted. In fundus albipunctatus andin Oguchi disease, the rod photoreceptors can achieve normal sensitivityto dim light but only after 2 or more hours of dark adaptation, comparedwith approximately 0.5 hours for normal individuals. Dryja, Am. J.Ophthalmol. 130:547, 2000. In each of these forms of stationary nightblindness, the poor rod sensitivity and the time course of darkadaptation correlate with the known or presumed physiologicabnormalities caused by the identified gene defects. Increasing theefficacy with which rhodopsin activates the phototransduction cascade isa possible new pharmacological approach to night blindness. Activatedrhodopsin activates the rod visual G protein, Gt, which activates thevisual transduction cascade. Pharmacologically increasing the effectivesignaling of rhodopsin can significantly impact people's ability to seeand function in low light. The ability, therefore, to identify smallmolecule compounds that enhance the ability of G protein coupledreceptors to signal would be a major benefit.

Because G proteins and their receptors influence a large number ofintracellular signals mediated by a large number of different chemicalligands, considerable potential for modulation of disease pathologyexists. Many medically significant biological processes are influencedby G protein signal transduction pathways and their downstream effectormolecules. See Holler et al., Cell. Mol. Life Sci. 340:1012-1020, 1999.G protein-coupled receptors and their ligands are the target for manypharmaceutical products and are the focus of intense drug discoveryefforts. Over the past 15 years, nearly 350 therapeutic agents targetingGPCRs have been successfully introduced into the market. Because of theubiquitous nature of G protein-mediated signaling systems and theirinfluence on a great number of pathologic states, it is highly desirableto find new methods of modulating these systems, including both agonistand antagonist effects. The ability to study the three-dimensionalconformations of GPCRs in response to different individual ligands withdifferent effects also is highly desirable, since these studies wouldaid in the search and development of drugs with particular structureswhich impart particular modulating effects on GPCRs.

Drug receptor theories are grounded in the law of mass action andinclude the concepts of affinity (the probability of the drug occupyinga receptor at any given instant), intrinsic efficacy (intrinsicactivity), which expresses the complex associations involving drug orligand concentration, and activation states of receptors. Drugsclassified as agonists interact with receptors to alter the proportionof activated receptors, thus modifying cellular activity. Conventionalagonists increase the proportion of activated receptors; inverseagonists reduce it. Direct agonists act on receptors, while indirectagonists facilitate the actions of the endogenous agonist (theneurotransmitter itself). Allosteric modulation of receptor activationis a new approach which circumvents the development of tolerance.

Most currently available drugs affecting GPCRs act by antagonizing thebinding between a G protein-coupled receptor and its extracellularligand(s). On the other hand, receptor subtype-selective drugs have beendifficult to obtain. An additional drawback to the classical approach ofdesigning drugs to interfere with ligand binding has been thatconventional antagonists are ineffective for some GPCRs such asproteinase activated receptors (PAR) due to the unique mechanism ofenzymatic cleavage of the receptor and generation of a tethered ligand.In other cases, intrinsic or constitutive activity of receptors leads topathology directly, thus rendering antagonism of ligand binding moot.For these reasons, alternative targets for blocking the consequences ofGPCR activation and signaling are highly desirable. Increasedunderstanding of the structural conformation of GPCRs under theinfluence of different agonists, antagonists or other ligands alsoallows design of compounds with highly specific effects on GPCRs.

One potential alternative target for inhibition by new pharmaceuticalshas been the receptor-G protein interface on the interior of the plasmamembrane. Konig et al., Proc. Natl. Acad. Sci. USA 86:6878-6882, 1989;Acharya et al., J. Biol. Chem. 272:6519-6524, 1997; Verrall et al., J.Biol. Chem. 272:6898-6902, 1997. The carboxyl terminus of Gα and otherregions of the G protein heterotrimer conform to a binding site at thecytoplasmic face of the receptor. Sondek et al., Nature 379:311-319,1996; Sondek et al., Nature 379:369-374, 1996; Wall et al., Science269:1405-1412, 1996; Mixon et al., Science 270:954-960, 1995; Lambrightet al., Nature 369:621-628, 1994; Lambright et al., Nature 379:311-319,1996; Sondek et al., Nature 379:369-374, 1996; Wall et al., Science269:1405-1412, 1996; Mixon et al., Science 270:954-960, 1995. Peptidescorresponding to these binding regions or mimicking these regions canblock receptor signaling or stabilize the active agonist-boundconformation of the receptor. Hamm et al., Science 241:832-835, 1988;Gilchrist et al., J. Biol. Chem. 273:14912-14919, 1998.

For example, in the case of rhodopsin, the rod photoreceptor, the GαC-terminal peptide, Gα 340-350, stabilizes the receptor in its activemetarhodopsin II conformation. Hamm et al., Science 241:832-835, 1988;Osawa and Weiss, J. Biol. Chem. 270:31052-31058, 1995. Two carboxylterminal peptides from GαS (354-372 and 384-394), but not thecorresponding peptides from Gαi₂, evoke high affinity agonist binding toβ₂-adrenergic receptors and inhibit their ability to activate Gαs andadenylyl cyclase. Rasenick et al., J. Biol. Chem. 269:21519-21525, 1994.Thus, the carboxyl terminus of Gα is important in mediating thespecificity of G protein responses. Drug discovery approaches which takeadvantage of this phenomenon, however, are not available. Jones et al.,Expert Opin. Ther. Patents 9(12):1641, 1999.

In general, GPCRs require agonist binding for activation. However, forsome receptors basic signaling activity may occur even in the absence ofan agonist (constitutive activity). In addition, modifications to thereceptor amino acid sequence can stabilize the active state conformationwithout the requirement for a ligand. Constitutive (agonist-independent)signaling activity has been demonstrated for both mutant and wild type(or native) form receptors (Tiberi and Caron, J. Biol. Chem.269:27925-27931, 1994; Hasegawa et al., J. Biol. Chem. 271:1857-1860,1996). A number of GPCRs that cause disease in humans, for example,receptors for thyroid-stimulating hormone (Vassart et al., Ann N.Y. AcadSci. 766:23-30, 1995), have been found to exhibit agonist-independentactivity. An inverse agonist is an agent that binds to the receptor andsuppresses this activity.

Experimentally, several single amino acid mutations have producedagonist-independent activity. β2 and α2 adrenergic receptors, forexample, mutated at single sites in the third cytoplasmic loop, showconstitutive activity. Ren et al., J. Biol. Chem. 268:16483-16487, 1993;Samama et al., Mol. Pharmacol. 45:390-394, 1994. In some cases, a largedeletion mutation in the carboxyl tail or in the intracellular loops ofGPCRs has led to constitutive activity. For example, in the thyrotropinreleasing hormone receptor a truncation deletion of the carboxylterminus or a smaller deletion in the second extracellular loop of thethrombin receptor renders the receptor constitutively active.Nussenzveig et al., J. Biol. Chem. 268:2389-2392, 1993; Matus-Leibovitchet al., J. Biol. Chem. 270:1041-1047, 1995; Nanevicz et al., J. Biol.Chem. 270:21619-21625, 1995.

These findings have led to a modification of traditional receptortheory. Samama et al., J. Biol. Chem. 268:4625-4636, 1993. It now isthought that receptors can exist in at least two conformations, aninactive conformation (R) and an activated conformation (R*), and thatan equilibrium exists between these two states that markedly favors Rover R* in the majority of receptors. It has been proposed that in somereceptors (native and mutant) there is a shift in equilibrium in theabsence of agonist that allows a sufficient number of receptors to be inthe active R* state to initiate signaling. Therefore, in response tochemical or physical external stimuli, GPCRs undergo a conformationalchange leading to the activation of heterotrimeric G proteins which goon to initiate intracellular signaling events.

Several studies suggest that many GPCRs exhibit properties consistentwith the existence of multiple conformational states. In rhodopsin, theexistence of multiple conformers is evident from absorbance changes.Sakmar, Prog. Nucleic Acid Res. Mol. Biol. 59:1-34, 1998. Activationoccurs by transition through intermediate conformations with theequilibrium between these forms showing a characteristic pH sensitivity.See Armis and Hoffman, Proc. Natl. Acad. Sci. USA 90:7849-7853, 1993;Vogel and Siebert, Biochemistry 41:3529-3535, 2002. Pharmacologicalstudies suggest that the existence of distinct receptor conformers canhave functional significance. Studies of fusion proteins of betaadrenergic receptor and G proteins suggest that partial agonistsstabilize a conformational state distinct from that stabilized by a fullagonist. Seifert et al., J. Pharmacol. Exp. Ther. 297:1218-1226, 2001.

The observation in several receptors that different agonists acting atthe same receptor can direct the relative activation of downstreampathways, a phenomenon called “signal trafficking,” also suggests thepresence of multiple populations of active receptor conformers. Kenakin,Trends Pharmacol. Sci. 16:232-238, 1995; Berg et al., Mol. Pharmacol.54:94-104, 1998; Cordeaux et al., J. Biol. Chem. 276:28667-28675, 2001;Marie et al., J. Biol. Chem. 276:41100-41111, 2001. Fluorescence studiesalso suggest the presence of different receptor conformationalpopulations when complexed with functionally distinct agonists. Ghanouniet al., J. Biol. Chem. 276:24433-24436, 2001. This emerging support forthe existence of distinct, functionally relevant conformers in severalGPCRs suggests that, for these receptors, the molecular activationmechanism must provide the means for switching among multipleconformations. A method to study these conformers by methods such ascrystallographic methods and NMR would be highly useful in the processof discovering compounds which can modulate or stabilize particularconformers.

Protein-protein interactions involved in regulatory phenomena arereversible and tend to involve only a small fraction of the proteinsurface. Generally, to identify peptides that block the protein-proteininteractions of interest particular peptides are synthesized in anattempt to mimic sections of one of the native interacting proteins oractive sequences are selected from random peptide libraries afterscreening. Peptides are made up of sequences of amino acids, howeverunlike DNA recognition, which is linearly coded into the sequence,peptide binding is dependent on three-dimensional structure.

The visual pigment, rhodopsin, is the most extensively studied member ofthe family of G protein receptors. Recently, the X-ray structure ofcrystalline bovine rhodopsin has been determined to a resolution of 2.8Å. This has paved the way for an understanding of the structure-functionrelationships of a prototypical GPCR at the molecular level. Sincerhodopsin constitutes greater than 90% of the disk membrane protein,measurements made on the proteins of disk membranes predominantlyreflect the properties of rhodopsin in its native environment. Rhodopsinconsists of the apoprotein opsin and the chromophore 11-cis retinal.Opsin, consisting of 348 amino acids, has a molecular mass of about 40kDa and folds into seven transmembrane helices of varying length and oneshort cytoplasmic helix. The retinylidene chromophore (the aldehyde ofvitamin A1) is covalently bound to Lys-296 in helix 7 via a protonatedSchiff base and keeps the receptor in an inactive conformation.

Light absorption causes a rapid 11-cis to all-trans isomerization of thechromophore which induces a series conformational of changes of theopsin moiety. This reaction occurs with high efficiency (quantum yield0.67) and the primary photoproduct, photorhodopsin, is formed within avery short time (200 fs). Subsequently, photorhodopsin thermally relaxeswithin a few picoseconds to a distorted all-trans configuration,bathorhodopsin. On a nanosecond time scale, bathorhodopsin establishesan equilibrium with a blue-shifted intermediate before the mixturedecays to form lumirhodopsin. Lumirhodopsin then is transformed intometarhodopsin I and subsequently metarhodopsin II, the activeconformation for G protein coupling. Thus, there are two conformationalswitches in rhodopsin which are controlled by the protonation ofspecific amino acids of the protein: the transition from the inactiveMeta I state to the active Meta II state and, in the absence of boundretinal, the transition from the inactive to the active state of opsin.According to current models, the receptor is kept in an inactiveconformation by electrostatic interactions between charged groups in theprotein, which are neutralized by the proton uptake involved in thetransition to an active state conformation.

The active receptor species Meta II decays slowly within minutes, byhydrolysis of the Schiff base and dissociation of the receptor into theapoprotein opsin and retinal. Researchers have shown that opsin is in apH-dependent conformational equilibrium between an active and aninactive state. During the decay of Meta II at neutral pH, moststructural changes of Meta II formation are reverted and the decayproduct opsin eventually adopts an active conformation similar to thatof Meta II.

Four distinct steps can be observed in the process of GPCR activation:(1) creation of the signal by a photon or by ligand binding; (2)transduction of the signal through the membrane; (3) interaction withthe G protein; and (4) activation of the second messenger. Although thephases clearly differ in the kind of processes taking place, they arenot discrete and independent. For example, allostery between ligandbinding and G protein binding has been observed for several GPCRs, aswell as cation-dependent allosteric regulation of agonist and antagonistbinding. Wessling-Resnick and Johnson, J. Biol. Chem. 262:12444-12447,1987; Hepler and Gilman, Trends Biol. Sci. 17:383-387, 1992; Nunnari etal., J. Biol. Chem. 262:12387-12392, 1987; Neve, Mol. Pharmacol.,39:570-578, 1991; Neve et al., Mol. Pharmacol. 39:733-739, 1991.

A number of cytoplasmic proteins interact exclusively withlight-activated rhodopsin (R*). Because the crystal structure depictsthe inactive form of rhodopsin as not interacting significantly withcytoplamic proteins, this structure can provide only indirectinformation about the R* state. In addition, two regions of thecytoplasmic surface domain of inactive rhodopsin structure (amino acidresidues 236-239 and 328-333) have not been fully resolved by crystalstructure analysis. Therefore, tools which can stabilize particularconformers would be useful for studying structure of GPCRs such asrhodopsin.

Negative antagonism is demonstrated when a drug binds to a receptor thatexhibits constitutive activity and reduces this activity. Negativeantagonists appear to act by constraining receptors in an inactivestate. Samama et al., Mol. Pharmacol. 45:390-394, 1994. Although firstdescribed in other receptor systems, negative antagonism has been shownto occur with GPCRs such as opioid, β2-adrenergic, serotonin type 2C,bradykinin, and D1B dopamine receptors. Schutz and Freissmuth, J. Biol.Chem. 267:8200-8206, 1992; Costa and Herz, Proc. Natl. Acad. Sci. USA86:7321-7325, 1989; Costa et al., Mol. Pharmacol. 41:549-560, 1992;Samama et al., Mol. Pharmacol. 45:390-394, 1994; Pei et al., Proc. Natl.Acad. Sci. USA 91:2699-2702, 1994; Chidiac et al., Mol. Pharmacol.45:490-499, 1994; Barker et al., J. Biol. Chem. 269:11687-11690, 1994;Leeb-Lundberg et al., J. Biol. Chem. 269: 25970-25973, 1994; Tiberi andCaron, J. Biol. Chem. 269: 27925-27931, 1994.

That being stated, the concept of constitutively active receptors offerinsights which explain pathophysiologic conditions. For example, aconstitutively active receptor in a disease process such as hypertensionmay no longer be under the influence of the sympathetic nervous system.In hypertension, a constitutively active GPCR may be expressed in anynumber of areas including the brain, kidneys or peripheral bloodvessels. A newly recognized class of drugs (negative antagonists orinverse agonists) which reduce undesirable constitutive activity can actas important new therapeutic agents. Thus, a technology for identifyingnegative antagonists (or understanding and stabilizing theconformational change in a GPCR that binding a negative antagonistcompound causes) of both native and mutated GPCRs has importantpredictable as well as not yet realized pharmaceutical applications.Furthermore, because at least some constitutively active GPCRs aretumorigenic, the identification of negative antagonists for these GPCRscan lead to the development of anti-tumor and/or anti-cell proliferationdrugs.

Mutagenesis studies of the carboxyl terminal region of Gαt haveidentified several specific amino acid residues in this binding regioncrucial for Gαt activation by rhodopsin. Martin et al., J. Biol. Chem.271:361-6, 1996. Substitution of three to five carboxyl-terminal aminoacids from Gαq with corresponding residues from Gαi allowed receptorswhich signal exclusively through Gαi subunits to activate the chimeric αsubunits and stimulate the Gαq effector, phospholipase C β. Conklin etal., Nature 363:274-276, 1993; Conklin et al., Mol. Pharmacol.50:885-890, 1996. All of these studies suggest that Gα carboxyl peptidesequences are responsible for the specificity of the signaling responsesof the individual G proteins. There are 16 unique Gα subunits (Gαi₁,Gαi₂, Gαi₃, GαO₁, GαO₂, GαZ, Gαt, Gαq, Gα11, Gα14, Gαs, Gα12, Gα13,Gα15/16, GαOLF and Gαgust) thought to mediate specific interaction withdifferent GPCRs, several hundred of which have been cloned. Thus,peptides corresponding to G protein regions which bind the GPCR could beused as competitive inhibitors of receptor-G protein interactions. Hammet al., Science 241-832-835, 1988; Gilchrist et al., J. Biol. Chem.273:14912-14919, 1998. Drug discovery approaches which take advantage ofthis opportunity, however, are not available. Jones et al., Expert Opin.Ther. Patents 9(12):1641-1654, 1999.

Identification of potent lead compounds for use in modern highthroughput screening assays and computerized design of new compoundsusing information about the desired three-dimensional conformation ofreceptor molecules, for example, are important aspects of the moderndrug discovery process. One of the major challenges confronting thoseusing these types of methods is the difficulty of identifying usefulbinding compounds from very large combinatorial libraries of potentialcandidate molecules. When literally hundreds of thousands of compoundsare screened, characterizing the compounds which test positive forbinding, for modulatory activity or for stabilization of a conformation(including false positives) is an expensive and time-consuming process.Hence, a method which can identify potent and useful lead compounds forhigh throughput screening and useful binding partners for threedimensional conformational studies and which reduce the number of falsepositives in the screening process would be very desirable.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a method of identifying a G proteincoupled receptor signaling modifying peptide, which comprises providinga peptide library based on a native G protein coupled receptor bindingpeptide; screening the peptide library for high affinity binding to theG protein coupled receptor; and selecting a member of the peptidelibrary having binding to the G protein coupled receptor of higheraffinity than that of the native peptide. The screening may be performedby testing for binding to an intact G protein coupled receptor or to atleast an intracellular fragment of a G protein coupled receptor.

The G protein coupled receptor binding peptide may be a G proteinsubunit or fragment thereof which is, for example from about 7 to about70 amino acids long or from about 7 to about 55 amino acids long or fromabout 8 to about 50 amino acids long or from about 9 to about 23 aminoacids long, and most preferably about 11 amino acids long. The G proteinsubunit fragment preferably is a Gα subunit or a Gα subunit carboxylterminal peptide but alternatively may be a Gβγ dimer.

Screening may comprise a competitive binding assay, which preferably ischaracterized by co-incubation of members of the peptide library withthe G protein coupled receptor binding peptide, for example in anenzyme-linked immunosorbant assay wherein the peptide library membersare capable of providing a detectable signal and/or wherein binding tothe G protein coupled receptor is determined by measuring a signalgenerated from interaction of an activating ligand with the G proteincoupled receptor.

The peptide library preferably is a combinatorial peptide library or aprotein-peptide fusion protein library such as, for example a peptidedisplay library or a maltose binding protein-peptide fusion proteinlibrary.

In another embodiment, the invention also provides a method ofidentifying a G protein coupled receptor signaling modifying compound,which comprises providing a library of candidate compounds to screen forbinding to the G protein coupled receptor; providing a high affinity Gprotein coupled receptor binding peptide; screening the library ofcandidate compounds for high affinity binding to the G protein coupledreceptor in competition with the high affinity G protein coupledreceptor binding peptide; and identifying a member of the library ofcandidate compounds having binding to the G protein coupled receptor ofequal or higher affinity than that of the high affinity G proteincoupled receptor binding peptide or a member of the library of candidatecompounds binding of which results in increased binding affinity of thehigh affinity G protein coupled receptor binding peptide. Screens may beperformed by testing for binding to an intact G protein coupled receptoror to at least an intracellular fragment of a G protein coupledreceptor.

The G protein coupled receptor binding peptide may be a G proteinsubunit or fragment thereof which is, for example from about 7 to about70 amino acids long or from about 7 to about 55 amino acids long or fromabout 8 to about 50 amino acids long or from about 9 to about 23 aminoacids long, and most preferably about 11 amino acids long. The G proteinsubunit fragment preferably is a Gα subunit or a Gα subunit carboxylterminal peptide but alternatively may be a Gβγ dimer.

Screening may comprise a competitive binding assay, which preferably ischaracterized by co-incubation of members of the peptide library withthe G protein coupled receptor binding peptide, for example in anenzyme-linked immunosorbant assay wherein the peptide library membersare capable of providing a detectable signal and/or wherein binding tothe G protein coupled receptor is determined by measuring a signalgenerated from interaction of an activating ligand with the G proteincoupled receptor.

The library of candidate compounds preferably is a focused library ofcandidate compounds based on the structure of the high affinity Gprotein coupled receptor binding peptide. The library of candidatecompounds may be a combinatorial library of, for example drug-likemolecules or a focused small molecule library whose members, for examplemay be based on the chemical structure of the high affinity G proteincoupled receptor binding peptide.

The invention also provides G protein coupled receptor signalingmodifying peptides and compounds identified according to the methodsdescribed above, as well as methods of modifying G protein coupledreceptor signaling in a cell having a G protein coupled receptor whichcomprise administering such compounds to the cell. Also provided aremethods of inhibiting G protein coupled receptor signaling whichcomprise contacting a compound with the G protein coupled receptor whichinterferes with binding of the G protein coupled receptor to its cognateG proteins.

In a further embodiment, the invention provides a method for identifyinga G protein coupled receptor signaling modifying compound, whichcomprises providing a peptide identified according to at least one ofthe methods described above, wherein the peptide is labeled to provide adetectable peptide signal; providing a library of candidate G proteincoupled receptor signaling modifying compounds; contacting the peptidewith the G protein coupled receptor under conditions such that thepeptide binds to the G protein coupled receptor; removing unboundpeptide from the G protein coupled receptor; measuring the signalingactivity of the peptide-bound G protein coupled receptor and measuringthe detectable peptide signal; contacting the members of the library ofcandidate G protein coupled receptor signaling modifying compounds withthe peptide-bound G protein coupled receptor; measuring the signalingactivity of the peptide bound G protein coupled receptor and measuringthe detectable peptide signal; determining whether the G protein coupledreceptor signaling activity is increased or decreased after contact withthe candidate compound and whether G protein coupled receptor peptidebinding is increased or decreased after contact with the candidatecompound; and identifying compounds for which contact with thepeptide-bound G protein coupled receptor results in both an increase inpeptide binding to the G protein coupled receptor and an increase in Gprotein coupled receptor signaling, identifying compounds for whichcontact with the peptide-bound G protein coupled receptor results inboth a decrease in peptide binding to the G protein coupled receptor anda decrease a G protein coupled receptor signaling and identifyingcompounds for which contact with the peptide-bound G-protein coupledreceptor results in increased binding affinity of the peptide identifiedaccording to a method described above. Methods for measuring thesignaling activity of the peptide-bound G protein coupled receptor maybe selected from the group consisting of measuring inositol phosphateaccumulation; measuring intracellular Ca²⁺ levels; measuring adenylcyclase activity; measuring transendothelial electrical resistance;measuring stress fiber formation; measuring ligand binding; measuringreceptor expression; measuring receptor desensitization; measuringkinase activity; measuring phosphatase activity; measuring nucleartranscription factors; measuring all migration (chemotaxis); measuringsuperoxide formation; measuring nitric oxide formation; measuring celldegranulation; measuring GIRK activity; measuring actin polymerization;measuring vasoconstriction; measuring cell permeability; measuringapoptosis; measuring cell differentiation; measuring membraneassociation of a protein that translocates upon GPCR activation, such asprotein kinase C; measuring cytosolic accumulation of a protein thattranslocates upon GPCR activation, such as protein kinase C; andmeasuring nuclear association of a protein that translocates upon GPCRactivation, such as Ran.

In yet a further embodiment, the invention provides compounds selectedfrom the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 13, 15, 17,21, 23, 25-27, 30, 32, 34, 36, 38, 40, 45-85, 94-111, 125-150, 160-164,175-178 and 183-264.

In yet a further embodiment, the invention provides a method forproviding a therapeutic G protein coupled receptor signaling modifierpeptide to a mammal which comprises administering to the mammal anexpression construct which expresses a compound selected from the groupconsisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 13, 15, 17, 21, 23, 25-27,30, 32, 34, 36, 38, 40, 45-85, 94-111, 125-150, 160-164, 175-178 and183-264. Further, the invention provides a method for treating a diseasestate in which excess G protein coupled receptor signaling is acausative factor, which comprises administering a compound selected fromthe group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 13, 15, 17, 21,23, 25-27, 30, 32, 34, 36, 38, 40, 45-85, 94-111, 125-150, 160-164,175-178 and 183-264.

In yet a further embodiment, the invention provides a method ofidentifying a G protein coupled receptor signaling enhancer, whichcomprises providing a peptide library based on a native G proteincoupled receptor binding peptide; screening the peptide library for highaffinity binding to the G protein coupled receptor; selecting a memberof the peptide library having binding to the G protein coupled receptorof higher affinity than that of the native peptide; providing a libraryof candidate compounds to screen for binding to the G protein coupledreceptor; screening the library of candidate compounds for high affinitybinding to the G protein coupled receptor in competition with a memberof the peptide library selected above; and identifying a member of thelibrary of candidate compounds having binding to the G protein coupledreceptor of equal or higher affinity than that of the peptide selectedabove or identifying a member of the library of candidate compoundsbinding of which results in increased binding affinity of the peptideselected above. Screening methods for use in this embodiment may includetesting for binding to an intact G protein coupled receptor or testingfor binding to at least an intracellular fragment of a G protein coupledreceptor. The G protein coupled receptor binding peptide may be a Gprotein subunit or fragment thereof, for example a G protein subunitfragment from about 7 to about 70 amino acids long, from about 7 toabout 55 amino acids long, from about 8 to about 50 amino acids long,from about 9 to about 23 amino acids long or most preferably about 11amino acids long. The G protein subunit fragment preferably is a Gαsubunit or a Gα subunit carboxyl terminal peptide but alternatively maybe a Gβγ dimer.

Screening may comprise a competitive binding assay, which preferably ischaracterized by co-incubation of members of the peptide library withthe G protein coupled receptor binding peptide, for example in anenzyme-linked immunosorbant assay wherein the peptide library membersare capable of providing a detectable signal and/or wherein binding tothe G protein coupled receptor is determined by measuring a signalgenerated from interaction of an activating ligand with the G proteincoupled receptor.

The library of candidate compounds preferably is a focused library ofcandidate compounds based on the structure of the high affinity Gprotein coupled receptor binding peptide. The library of candidatecompounds may be a combinatorial library of, for example drug-likemolecules or a focused small molecule library whose members, for examplemay be based on the chemical structure of the high affinity G proteincoupled receptor binding peptide.

Enzyme-linked immunosorbant assays for use in the inventive method maycomprise the steps of immobilizing the G protein coupled receptor onto asolid support; providing a protein-peptide fusion protein displaylibrary; incubating members of the protein-peptide fusion proteindisplay library with the immobilized G protein coupled receptor in thepresence of the G protein coupled receptor binding peptide underconditions such that members of protein-peptide fusion protein displaylibrary having a binding affinity for the G protein coupled receptor atleast as high as the G protein coupled receptor binding peptide bind tothe immobilized G protein coupled receptor; removing unbound members ofthe protein-peptide fusion protein display library; incubating the boundprotein-peptide fusion protein display library with antibodies whichspecifically recognize the protein portion of the protein-peptide fusionprotein display library members under conditions such that theantibodies specifically bind to the protein-peptide fusion proteindisplay library members; removing unbound antibodies; and detecting thebound antibodies. The protein-peptide fusion protein display librarypreferably is a maltose binding protein-peptide fusion protein displaylibrary and the antibodies preferably are anti-maltose binding proteinantibodies. Binding to the G protein coupled receptor preferably isdetermined by measuring a signal generated from interaction of thesignalling enhancer with the G protein coupled receptor.

The peptide library preferably is a combinatorial peptide library, forexample a protein-peptide fusion protein library such as a maltosebinding protein-peptide fusion protein library or any suitable peptidedisplay library. Libraries of candidate compounds preferably are focusedlibraries of candidate compounds based on the structure of the compoundselected above as having binding to the G protein coupled receptor ofhigher affinity than that of the native peptide. The library may be apeptide library or a small molecule library.

In yet a further embodiment, the invention provides compounds identifiedby a method as described above. In yet further embodiments, theinvention provides a method for treating a disease state in whichalterations in G protein coupled receptor signaling is a causativefactor and a method for treating a disease state in which alterations inG protein coupled receptor signaling is a causative factor both of whichcomprise administering these compounds. In yet a further embodiment theinvention provides a method of determining the three-dimensionalstructure of a G protein coupled receptor, which comprises contactingthe G protein coupled receptor with a compound identified by at leastone of the methods described above under conditions such that bindingoccurs and a conformation of the G protein coupled receptor isstabilized; co-crystallizing the G protein coupled receptor-compoundbinding pair; subjecting the co-crystallized binding pair to X-raycrystallography; and determining the three-dimensional structure of theco-crystallized binding pair, wherein atomic coordinates of the Gprotein coupled receptor are obtained. In yet a further embodiment, theinvention provides a method of determining the three-dimensionalstructure of a G protein coupled receptor, which comprises contactingthe G protein coupled receptor with a compound identified by at leastone of the methods described above under conditions such that bindingoccurs and a conformation of the G protein coupled receptor isstabilized; subjecting the binding pair to nuclear magnetic resonancestudy; and determining the three-dimensional structure of the bindingpair, wherein atomic coordinates of the G protein coupled receptor areobtained.

In yet a further embodiment, the invention provides a method ofisolating a G protein coupled receptor binding partner, which comprisesproviding a solid support comprising bound compound identified by atleast one of the methods described above; providing a library ofcandidate G protein coupled receptor binding partner compounds;contacting the library of candidate compounds with the solid supportunder conditions such that binding of the candidate compounds to thecompound occurs; eluting unbound and nonspecifically bound candidatecompounds from the solid support; and recovering bound candidatecompounds from the solid support.

In yet a further embodiment, the invention provides a method ofdesigning small molecules that modify activation of a G protein coupledreceptor, which comprises determining the three-dimensional structure ofa G protein coupled receptor according to at least one of the methodsdescribed above; and designing candidate structures by computer modelingbased on the atomic coordinates, wherein the candidate structures arepredicted to bind to the G protein coupled receptor.

In yet a further embodiment, the invention provides a nucleic acid whichcomprises a DNA that encodes a peptide identified by at least one of themethods described above, wherein the DNA is operably linked to aheterologous transcriptional regulatory sequence, an expression vectorwhich comprises this nucleic acid and a cell transfected with theexpression vector. The invention also provides an antibody thatspecifically recognizes a peptide identified by any of the methodsdescribed above, such as, for example, a monoclonal antibody, apolyclonal antibody, a humanized antibody or a single chain antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C are schematic diagrams showing the basic two-stepplatform.

FIGS. 2A and 2B are schematic diagrams showing an example of the basisfor the affinity screening method used to separate and identify GPCRbinding peptides.

FIG. 3 is a schematic diagram of vector pJS142.

FIG. 4 is a schematic diagram showing an ELISA procedure.

FIG. 5 provides results showing that the LacI-Gq fusion protein bindsthrombin receptor in a concentration-dependent manner.

FIG. 6 shows data from binding assays performed on some of the clonesidentified using peptide 8 in the screening process.

FIG. 7 provides binding data for LacI peptide fusion proteins to PAR1receptor. pELM6 is the MBP vector alone; pELM17 is the MBP-nativeGt340-350 peptide fusion protein.

FIG. 8 is a bar graph comparing binding of high affinity fusion proteinsto the high affinity peptide 8 fusion protein (MBP 8).

FIG. 9 presents ELISA results from panning CHO cells overexpressinghuman thrombin receptor (PAR1) using purified MBP-C-terminal fusionproteins. MBP-G11=xxxx (SEQ ID NO: 1) LQLNLKEYNLV (SEQ ID NO: 2);PAR-13=VRPS (SEQ ID NO: 3) LQLNRNEYYLV (SEQ ID NO: 4); PAR-23=LSRS (SEQID NO: 5) LQQKLKEYSLV (SEQ ID NO:6); PAR-33=LSTN (SEQ ID NO: 7)LHLNLKEYNLV (SEQ ID NO: 8); PAR-34=LPQM (SEQ ID NO: 9) QRLNVGEYNLV (SEQID NO: 10); PAR-45=SRHT (SEQ ID NO: 11) LRLNGKELNLV (SEQ ID NO:12).

FIG. 10 presents a dose-response curve of SF9 membranes (PAR1 receptor)assayed with lacI-Gq lysates.

FIG. 11 is a concentration response curve demonstrating binding ofnative Gq peptide-maltose binding protein to PAR1 reconstituted in lipidvesicles.

FIG. 12B is a schematic diagram showing an exemplary cDNA minigeneconstruct. SEQ ID NOS:270 and 271 are shown in FIG. 12A.

FIG. 13 shows an agarose gel of a NcoI digest of minigene vector. Lane 1is a 1 kb DNA ladder; lane 2 is pcDNA 3.1; lane 3 is pcDNA-Gαi; lane 4is pcDNA-GαiR; and lane 5 is pcDNA-Gαq.

FIG. 14 shows an agarose gel of PCR products showing transcription ofpeptide minigene RNA in transfected cells. Lane 1 contains size markers,lane 2 contains PCR products from cells transfected with pcDNA-GiR, lane3 contains PCR products from cells transfected with pcDNA-Gi, and lane 4contains PCR products from cells transfected with pcDNA3.1, the emptyvector.

FIG. 15 is a bar graph showing the relative [³H] inositol phosphateproduction after thrombin stimulation normalized against the basalvalue.

FIG. 16 presents data showing inhibition of a GPCR mediated increase inintracellular calcium concentration in the presence or absence of aminigene vector encoding the identified high affinity peptide. FIG. 16Apresents fluorescence ([Ca⁺⁺]_(i) level) increase 30 seconds afterthrombin addition. FIG. 16B shows the kinetics of [Ca⁺⁺] fluorescencechanges after cell stimulation with thrombin.

FIG. 17 presents data showing inhibition of a GPCR-mediatedphosphoinositol (P1) hydrolysis in the presence or absence of a minigenevector encoding the identified high affinity peptide.

FIG. 18 is a bar graph indicating relative GPCR-mediated increase ofMAPK activity in the presence or absence of a minigene vector encodingthe identified high affinity peptide in cells expressing GPCR-bindingpeptides.

FIG. 19 shows reduction of thrombin-induced transendothelial electricalresistance in cells expressing Gαq, Gαi, GαiR or empty vector.

FIGS. 20A, 20B, 20C and 20D are a series of photographs of cells stainedfor F-actin, showing the inhibition of stress fiber formation afterexposure to thrombin in cells expressing pcDNA-G12 or pcDNA-G13 minigeneconstruct.

FIGS. 21A, 21B and 21C are bar graphs showing acetylcholine (Ach)response (pA/pF) for HEK 293 cells transiently transfected withGIRK1/GIRK4 and the indicated minigene construct.

FIG. 22 demonstrates selective G protein mediated adenylyl cyclaseinhibition in cells expressing minigene constructs containing Gαcarboxyl terminal peptide inserts.

FIG. 23 presents dose-response curves of MII stabilization by αt340350,mutant αt340-350K341L and heterotrimeric Gt.

FIG. 24 shows stabilization of MII by small molecule PL_(—)0302R3C4.

FIG. 25 presents fluorescence data showing super agonists for rhodopsinhave no effect on PAR1-stimulated Ca²⁺ transients.

FIGS. 26A and 26B are graphs showing light responses (as measured by achange in current) from isolated rods of dark-adapted salamander retinasin the presence of small molecule PL_(—)0302R3C4.

FIGS. 27A and 27B are graphs showing light responses (as measured by achange in current) from isolated rods of dark-adapted salamander retinasin the absence of a small molecule.

FIG. 28B is an MBP-8 binding curve with added compound PL_(—)1012R2C1,the structure of which is depicted in FIG. 28A, showing the compound'sability to enhance MBP-8 binding to EDTA-washed rhodopsin.

FIG. 29B is an MBP-8 binding curve with added compound PL_(—)0894R3C7,the structure of which is depicted in FIG. 29A, showing the compound'sability to enhance MBP-8 binding to EDTA-washed rhodopsin.

FIG. 30B is an MBP-8 binding curve with added compound PL_(—)0568R1C5,the structure of which is depicted in FIG. 30A, showing the compound'sability to enhance MBP-8 binding to EDTA-washed rhodopsin.

FIG. 31B is an MBP-8 binding curve with added compound PL_(—)0551R8C1,the structure of which is depicted in FIG. 31A, showing the compound'sability to enhance MBP-8 binding to EDTA-washed rhodopsin.

FIG. 32B is an MBP-8 binding curve with added compound PL_(—)0302R3C4,the structure of which is depicted in FIG. 32A, showing the compound'sability to enhance MBP-8 binding to EDTA-washed rhodopsin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention involves a method of identifying compounds whichcan interfere with or increase binding between a G protein-coupledreceptor (GPCR) and its cognate G protein(s) and compounds whichstabilize a particular conformation of a GPCR for conformational study.These compounds modulate G protein-mediated signaling and thus can beused as pharmaceuticals, as lead compounds for identification ofpotential useful drugs, as components of assays which identify drugcandidates or as binding partners in conformational studies by knownmethods, such as for example X-ray crystallography or nuclear magneticresonance.

Methods for screening and drug identification use peptides that mimicthe structure of the GPCR binding regions of G proteins and thus areable to modulate receptor-G protein interactions specifically orspecifically bind to a given receptor with high affinity. These highaffinity peptides can be delivered into cells in the context of anexpression construct to act as blockers or agonists of specificreceptor-mediated cellular responses in vitro and in vivo or can beadministered directly to a patient. The peptides also form the basis ofa screening, identification and selection process to provide traditionalpharmaceutical compounds or to study structure-function relationships inbinding. In particular, the invention allows one to identify highaffinity analog peptides that block or mimic compounds at the receptor-Gprotein interface for a particular G protein and to use these highaffinity analogs in a high throughput screen to identify other peptidesor small molecules that likewise specifically antagonize or agonize GPCRsignaling for a G protein or class of G proteins.

Small molecules can be used in analogous high throughput screeningprocesses to identify further compounds. “Small molecule” denotes anynon-peptide organic compound which binds or interferes with binding tothe interfacial region of a GPCR or is a candidate for such action. Suchmolecules that bind to and stabilize a particular conformer of a GPCRalso are included in the definition of “small molecule” as used herein.Peptides or small molecules directed at the receptor-G protein interfacecan be designed using the inventive method to inhibit or enhancebiological processes that employ signaling through a GPCR or to bind toand stabilize a particular GPCR conformer. Such compounds which bind to,interfere into binding to or stabilize a conformer of the GPCR-G proteininterface (including but not limited to agonists, inverse agonists,allosteric agonists, blockers, antagonists, inhibitors, negativeantagonists, partial agonists, and enhancers, as well as compounds whichbind to and stabilize a particular conformer) are termed “modifiers” or“modifying” compounds, and may include both peptides and smallmolecules. This approach to drug design is useful in targeting Gprotein-GPCR interactions for which there are no available ligands,orphan receptors the ligands of which are not known, mutantconstitutively activated receptors, antibody-crosslinked irreversiblyactivated receptors such as TSH receptors in Graves' Disease, andproteinase activated receptors (PAR). It works equally well, however,with any GPCR-G protein interaction and more broadly, withreceptor-protein interactions in general.

Because the method is useful for identifying high affinity compoundsthat can bind to and enhance or inhibit virtually any GPCR, the approachis useful in identifying compounds which can prevent, ameliorate orcorrect dysfunctions or diseases in which a specific class of G proteinsis relevant. Conditions and disease states for which GPCR enhancers andinhibitors are useful include, but are not limited to: stroke;myocardial infarction; restenosis; atherosclerosis; hypotension;hypertension; angina pectoris; acute heart failure; cardiomyocyteapoptosis; cancers; infections such as bacterial, fungal, protozoan andviral infections, and particularly infections caused by HIV-1 or HIV-2;septic shock; pain; chronic allergic disorders; asthma; inflammatorybowel disease; osteoporosis; rheumatoid arthritis; Graves' disease;post-operative ileus; urinary retention; testotoxicosis; ulcers;obesity; benign prostatic hypertrophy; and psychotic and neurologicaldisorders including anxiety, epilepsy, schizophrenia, manic depression,Parkinson's disease, Alzheimer's disease, delirium, dementia, drugaddiction, anorexia, bulimia, mood disorders and sleep disorders;smoking cessation and any other disease or condition that can be treatedby G protein coupled receptor inhibition and any other disease orcondition that can be treated by G protein coupled receptor activation.Treatment of this diverse set of disorders is possible because thereceptors to which various G proteins bind differ enough to allow thecreation of a battery of analog peptides which can specificallyinterface with different GPCR or different classes or groups of GPCR.The relationship of G proteins and G protein signalling to variousdiseases and conditions such as those listed above is known in the art.

With the inventive screening methods, the sequences identified in aparticular screen do not bind to all receptors, but only to theparticular receptor of interest. The interaction between a G protein anda GPCR is quite specific. For example, a difference in one amino acidcan substantially reduce or eliminate the ability of the Gαi_(1/2)peptide to bind the A1 adenosine G protein coupled receptor-G proteininterface. Gilchrist et al., J. Biol. Chem. 273:14912-14919, 1998. Bothupstream regulation of GTP/GDP exchange on G proteins and Gprotein-mediated effector activation may be inhibited with interfacialbinding compounds. Thus, high affinity analog peptides can be designedto specifically interfere with or stabilize a particular action of oneGPCR. Likewise agonizing or enhancing peptides also specifically affectone GPCR. These specifically-acting peptide analogs are useful both aspharmaceutical compounds per se, and as potent lead compounds in modernhigh throughput screens for other peptides and small molecule bindershaving the same specific GPCR interaction.

The inventive methods, in one embodiment, rely as first step onscreening for small molecules that enhance or inhibit the ability of thereceptor to interact with the heterotrimeric G protein. Using rhodopsinand transducin, the screen has found small molecules that significantlyenhance rhodopsin's ability to form MII, the active form of rhodopsin.Such small molecules can serve as lead compounds in drug discoveryefforts directed towards potential therapeutic agents to combat nightblindness. Using the screen to identify small molecules, and thentesting the identified compounds using in vitro and in vivo analysiswill result in discovery of potent, high affinity compounds.

This invention therefore can be used to identify small molecules thatenhance the ability of rhodopsin to signal. The inventive methodsinvolve, in one embodiment, screening compound libraries to discovermore molecules that increase binding of G protein peptides to activatedrhodopsin. The methods also include testing these molecules in a numberof assays to determine their effects on rhodopsin signaling, includingMII stabilization, guanosine 5′-O-(3-[³⁵S]thio) triphosphate (GTPγS)binding, and 3′,5′ cyclic GMP phosphodiesterase (PDE) activation.

Additionally, the methods involve testing the small molecules for theirspecificity by measuring their effects using another GPCR, for example,human thrombin receptor (PAR1), which also has been shown to couple toGt. Seibert et al., Vision Res. 42:517,1999. Enhancement of thesensitivity of vision in vivo can be tested according to a method of theinvention using electroretinography (ERG) of wild type and mutant mice.Chang et al., Vision Res. 42:517-525, 2002. The inventive methods alsooptionally involve optimizing the chemical structure of enhancers orantagonists, performing pharmacokinetic, toxicological and metabolismstudies of the discovered chemical entities, and large animal efficacystudies, and clinical trials for a pharmacological treatment for nightblindness. Therefore, the methods of the invention can be used, forexample, to identify small molecules that enhance the binding of thehigh affinity Gt peptides to light activated rhodopsin, determinewhether the small molecules enhance rhodopsin signaling in vitro,determine if the small molecules are specific for rhodopsin, or if theycan enhance other GPCR-G protein signaling events, and test the smallmolecules in a mouse model for stationary night blindness for increasedsensitivity of vision as measured by ERG.

A first step was to identify peptides with sequences based on theC-terminus of Gt that bind with high affinity to either light-activatedor dark-adapted rhodopsin. These peptide analogs were then tested fortheir specificity to binding to rhodopsin versus other GPCRs, as well astheir ability to stabilize the MII conformation of the receptor. Themethods of the invention also identify small molecules that bindlight-activated rhodopsin and by doing so enhance the binding of thehigh affinity peptide analogs. The binding affinity (EC₅₀) of thecompound is the first criterion of a successful drug candidate. Theidentified small molecules are tested in vitro for their ability toenhance rhodopsin signaling using assays such as MII stabilization andMII decay, GTPγS binding, and PDE activation.

Upon activation, rhodopsin undergoes a conformational change that allowsits interaction with and activation of Gt, leading ultimately to thestimulation of PDE. The binding of Gt to light-activated rhodopsininduces a high affinity receptor state that can be measuredspectrophotometrically by stabilization of the active, signalingmetarhodopsin II state of the receptor. Using a split-beam SLM AmincoDW2000 spectrophotometer, for example, one can determine if the receptorundergoes proper conformational changes following light activation. Thisassay shows the small molecule acting on the conformation of thereceptor. If rhodopsin's active intermediate, metarhodopsin II (MII), isstabilized by the presence of the small molecule, the activation energyof the receptor is lowered. Using the inventive assay system, compoundswere identified that allow the receptor to enter the active, MIIconformation without hetereotrimeric G protein, which normally isrequired. The “enhancers” stabilize the active (signaling) conformationof the receptor. “Inhibitors” block the binding of transducin torhodopsin and thus inhibit the receptor from entering the properconformation even in the presence of agonist (light) and G protein.

Metarhodopsin II decay can be used to examine the differences incompound potencies are due to changes in MII decay. It could bepostulated that differences could be due to effects of a compounds tonon-specifically attach the retinal Schiff's base linkage of MII. Thus,one can compare the time dependent MII decay in the presence of theindividual compounds. In the process of receptor activation, the Gαtsubunit binds a GTP molecule. GTPγS binding assays can determine theability of the receptors to signal, with an increase in GTPγS bindingindicating receptor-mediated release of the GDP from the α subunit andsubsequent binding of GTP. Conversion of inactive transducin (Gt•GDP) tothe active state (Gt•GDP) is accompanied by dissociation of the Gα fromGBγ. The free Gαt•GTP then activates cGMP phosphodiesterase (PDE) bybinding to and dissociating its two inhibitory γ subunits. As a result,the released catalyzing α and β subunits of activated PDE (PDE*) canconvert cGMP to GMP. Therefore, compounds which affect rhodopsinsignaling can be tested for their affects on PDE assays.

Generally, small molecules that display an appropriate dose curve whenused to compete off the high affinity peptide fusion proteins, with aresulting EC₅₀<100 μM for binding to rhodopsin are suitable forcontinued study and are tested for the ability to stabilize MII.Preferably, those with an EC₅₀<10 μM for MII stabilization are analyzedfurther. Further analysis may include thermal stability of rhodopsin inthe presence of the small molecules (MII decay), and GTPγS binding (anassay for the small molecule's effects on function). The rate ofGTPγS-binding is controlled by a rate-limiting GDP release of Gαsubunits. Native Gαt, in the presence or absence of Gβγt, displays veryslow intrinsic rates of GDP release. Therefore, an increase in guanosine5′-O-(3-[³⁵S]thio) triphosphate(GTPγS) binding indicates receptormediated release of the GDP from the α subunit and subsequent binding ofthe GTP.

GTPγS binding assays may be performed as follows or using any methodknown in the art. Gα subunits (1 mM) alone, or Gα subunits in thepresence of the small molecules to be assayed are mixed with 2 mM Gβγtor 2 mm Gβγt and urea-washed ROS membranes (500 nM rhodopsin) andincubated for 3 min at 25° C. Binding reactions may be started byaddition of 5 mm [³⁵S]GTPγS (0.1 mCi). Aliquots of 50 μl are withdrawnat several timepoints, mixed with 1 ml ice-cold 20 mm Tris.2HCl (pH 8.0)buffer containing 130 mM NaCl and 10 mM MgSO₄ and passed throughWhatman™ cellulose nitrate filters (0.45 mm). The filters are washedthree times with the same buffer (3 ml, ice-cold) and counted in aliquid scintillation counter after dissolution in 3a70B mixture. SeeSkiba et al., J. Biol. Chem. 271: 413-424, 1996 for exemplary methodswhich may be used with the invention. The skilled person will recognizevariations and adjustments which may be made to the assay, and suchvariations are considered within the scope of this invention. Thek_(app) values for the binding reactions may be calculated by fittingthe data to the equation, GTPγS bound (% bound)=100% (1−e^(−kt)). Thesmall molecule(s) also may be tested for the ability to affect PDEactivation. Gtα binding to PDEy relieves the inhibitory effect of thegamma subunit on the catalytic α and β subunits of PDE and allows thehydrolytic activity of these subunits to be increased almost 300 fold.

Activation of Gtα by rhodopsin can be monitored in the presence orabsence of the small molecules using fluorescence spectroscopy at 20° C.as described by Cerione, Methods Enzymol. 237:409-423, 1994. This assaymeasures the Gα:GTPγS (complex between a subunit of transducin andGTPγS) formation rate catalyzed by wild-type rhodopsin uponillumination. The excitation wavelength is 295 nm (2 nm bandwidth), andfluorescence emission is monitored at 340 nm (12 nm bandwidth). Briefly,rhodopsin (40 nM) is added to a solution of Gt (250 nM) in a reactionmixture containing 10 mM Tris (pH 7.2), 2 mM MgCl₂, 100 mM NaCl, 1 mMDTT, and 0.01% n-dodecyl β-maltoside. The solution is stirred for 300sec to equilibrate. GTPγS (5 μM) is added to the reaction mixture to afinal concentration of 5 μM, and the increase in fluorescence isfollowed for and additional 2000 sec. To calculate the activation rates,the slopes of the initial fluorescence increase after GTPγS additionwere determined through the data points covering the first 60 sec. Thevalues in the presence of the small molecules may be normalized to thevalue obtained for wild-type rhodopsin with no compounds taken as 1.00.Those molecules which appear to be acting directly on rhodopsin in theseassays, or variations on these assays readily apparent to the person ofskill in the art are taken to the next level of testing. The smallmolecules also are assayed for the ability to modulaterhodopsin-transducin signaling specifically without affecting processesmediated by other GPCRs.

Preferred small molecule “enhancers” and “inhibitors” are uniquelyspecific, not only for the receptor, but for the receptor-G proteininteraction. As there are over 1000 GPCRs, and no simple way todetermine the effect of compounds on each and every one of themindividually, a few select and representative GPCR signaling systems maybe tested. Functional coupling of the human thrombin receptor (PAR1)with Gt has been demonstrated. Seibert, Eur. J. Biochem. 266(3):911-916,1999. Testing for effects on PAR1 may include determining if the smallmolecule(s) have an effect on thrombin-mediated signal transductionevents such as adenylyl cyclase activity, calcium influx, and inositolphosphate accumulation. Other tests for functional coupling to PAR1 orother GPCRs are known in the art and may be used as well.

Adenylyl cyclase activity may be measured in a final volume of 50 μlwith [α-³²P]ATP (1 mM; 120-400 cpm/pmol) as the substrate and[2,8-³H]cAMP (2 mM; 200,000 cpm/pmol) to monitor recovery in an assaymixture containing 5 mM MgCl₂, 1 mM EDTA, 1 mM 2-mercaptoethanol, 100 μMpapaverine, 1 pg/ml bovine serum albumin, and an ATP-regenerating systemconsisting of 20 mM creatine phosphate and 120 units creatinephosphokinase/ml in 25 mM Tris-HCl buffer, pH 7.5. The concentration ofATP and cAMP may be determined spectrophotometrically at 259 nm, using εvalues of 15.4 and 14.6 mM¹cm⁻¹, respectively. The assay may beinitiated by addition of protein and after a 10 min incubation at 37° C.the reaction is stopped with 2 volumes stop solution (2% SDS/45 mMATP/13 mM cAMP). The samples then may be heated (e.g. to 100° C. for 3minutes) and the formed cyclic [α-³²P]AMP recovered. See Gilchrist etal., J. Biol. Chem . 276:25672-25679, 2001, the disclosures of which arehereby incorporated by reference. Finally, the compounds passing theprevious steps may be tested in an animal model of night blindness asdescribed by Chang et al., Vision Res. 42:517-525, 2002, the disclosuresof which are hereby incorporated by reference.

To assess the effects of small molecules on photoresponses in an in vivosystem, electroretinography of mice exposed to the small molecule(s) maybe used to measure the amplitude of both the a- and b-waves. Plots ofthe amplitude against the logarithm of relative light intensitiesindicate if the compounds are affecting only rod signaling. Thesensitivity for eliciting a threshold b wave within normal limits alsomay be measured. Mutant animals also may be tested to observe not onlythe effects of enhancers on wild type rhodopsin signaling, but also theeffects on animals with night blindness.

The small molecules to be tested may be dissolved in sterile PBS andadministered as eye drops. Experiments may be repeated using IV or IMinjections if initial results are negative. The most promisingcandidates undergo the steps needed to take them from an identifiedcompound to a lead compound. This approach identifies a pharmacologicaltreatment for night blindness which circumvents the need for moreinvasive procedures such as gene therapy, laser ablation and retinalreplacement.

Mapping the sites of interaction between proteins involves identifyingparts of the interface between two proteins using synthetic peptidescorresponding to interfacial regions. The peptides are identifiedbecause they act as competitive inhibitors of the interaction. NMRstudies of peptide structures in their bound conformation using trNOESY,combined with analysis of activity of substituted peptide analogs todefine the minimal structural requirements for interaction were used tounderstand the structural basis of rhodopsin-transducin interaction aswell as G protein-effector interaction. Peptides corresponding to theC-terminus of Gt can be used to stabilize rhodopsin in its activeconformation (MII) or in an inactive conformation. The 3-dimensionalstructures of heterotrimeric G proteins reveals that the last 7 aminoacids of G protein α subunits are unstructured, indicating that thisregion of the α subunit is critical for binding to the cytoplasmicsurface of an activated receptor with induced fit. This interaction isquite specific since a difference in a single amino acid can affect theaffinity by 1000 fold.

High throughput screening is a recent technology that has been developedprimarily within the pharmaceutical industry. It has emerged in responseto the profusion of new biological targets and the need of thepharmaceutical industry to generate novel drugs rapidly in a changedcommercial environment. Its development has been aided by the inventionof new instrumentation, by new assay procedures, and by the availabilityof databases that allow huge numbers of data points to be managedeffectively. High throughput screening combined with combinatorialchemistry, rational design, and automation of laboratory procedures hasled to a significantly accelerated drug discovery process compared tothe traditional one-compound-at-a-time approach. Screens may beperformed manually, however robotic screening of the compound librariesis preferred as a time- and labor-saving device.

One critical aspect of the drug discovery process is the identificationof potent lead compounds. A purely random selection of compounds fortesting is unlikely to yield many active compounds against a givenreceptor. Typically, pharmaceutical companies screen 100,000 or morecompounds per screen to identify approximately 100 potential leadcompounds. On average, only one or two of these compounds actuallyproduce lead compound series. Therefore, companies have been assayinglarger and larger data sets in the search for useful compounds. Compoundaccessibility then becomes an issue: historical compound collections arelimited in size and availability. In contrast, large combinatorialchemistry libraries can be synthesized on demand, but at significanttechnical difficulty and cost. As the library sizes expand, thedifficulty becomes selecting the desired compounds from these very largecombinatorial libraries. When literally hundreds of thousands ofcompounds are screened, it makes characterizing the candidate leadcompounds an expensive and time-consuming process, particularly whenmany of the “hits” turn out to be false positives.

The multi-step approach to the drug discovery process described hereprovides a solution to many of these problems. One embodiment of thisinvention takes advantage of the properties of G protein α subunitcarboxyl termini to identify peptides which act as high affinity,competitive inhibitors or agonists of G protein/GPCR interactions. Themethod, however, can be used with any specific protein-protein,protein-small molecule, protein-nucleic acid interaction or the like. Inaddition, peptides based on any region of a Gα subunit or any region ofa Gβγ dimer which is involved in GPCR binding may be used in the sameway. Many such GPCR binding regions are known in the art. Theidentification of high affinity competitors forms a first step in ascreening and selection method which overcomes many of the disadvantagesof high throughput screening by providing specific, high affinity leadcompounds against which to test potentially useful pharmaceuticals.Because peptides selected by this method have affinity for their bindingpartner up to 1,000 times higher or more than the native protein, thisstep is one key to successfully screening and identifying usefulpharmaceutical compounds.

A subsequent step of the preferred process involves high throughputscreening of candidate peptide or small molecule pharmaceuticalcompounds against the high affinity lead peptides identified in thefirst step. Because the lead peptide compounds are potent and specificbinders to the desired receptor, screening assays test for compoundsthat can decrease the binding of the peptide (“blockers”) or thatincrease the binding of the peptide (“agonists”). The assay systemallows one to measure both binding and function simultaneously as thepeptides all serve to mimic a required step, that of specific receptor-Gprotein binding. By using this site, the system facilitatesidentification of those candidate compounds which bind not only withuseful affinities (nM to μM range) but by the very virtue of theirselection process will affect function by either increasing ordecreasing the G protein binding. The high throughput screening step ofthe drug discovery process is thereby greatly simplified, because thenumber of false positive compounds, and compounds which are identifiedas binders but which bind only with low affinity, is reduced orvirtually eliminated. Only those compounds with a high chance of successwill be identified by the screen, therefore useful compounds can beidentified directly and there are many fewer compounds which need to becharacterized and further studied to confirm that the compounds arespecific, potent pharmaceutical compounds. In addition, the methodidentifies a compound through binding directly to the precise site ofinterest, so that the mechanism of binding and the mechanism of actionof the newly identified pharmaceutical compound does not have to bediscovered and confirmed later by a separate process.

The identified high affinity peptides also may be used according to theinventions to identify GPCR inverse agonists. High affinity peptidesidentified in a first step of the inventive method bind the receptor andstabilize it in an active or R* conformation. Screens which are used toidentify potent agonists seek out compounds which can compete with thisbinding and also stabilize the GPCR in its R* state. Inverse agonists,on the other hand, stabilize the GPCR in an inactive or R state.Therefore, screens designed to detect dissociation of the high affinitypeptide or a decrease in its affinity for the GPCR are used to identifyinverse agonists.

Although this description provides examples relative to the interactionbetween a G protein coupled receptor and its cognate Gα protein, themethodology can be used to identify peptide inhibitors of mostprotein-protein interactions, specifically including any interactionbetween a GPCR and any region of a Gα or Gαγ G protein subunit. The highaffinity peptides selected by this method may be used in high throughputscreening to identify small molecules that can be used as modulators ofa variety of specific biological process.

To produce very high affinity peptide GPCR blockers, the tertiarystructure of a wild-type Gα carboxyl terminal peptide or any other GPCRbinding peptide in its receptor-bound conformation may be studied, forexample, using trNOESY (NMR). Dratz et al., Nature 363:276-280, 1993.Structural data derived from these types of studies of G protein regionsare combined with analysis of activity of substituted peptide analogs todefine the minimal structural requirements for interaction of peptidesor any ligand with GPCR. The following experimental systems are examplesof systems which can be used to define receptor-G protein interactions:(i) rhodopsin-transducin (Gαt) in retinal rod cells, (ii) β-adrenergicreceptor-Gαs in C6 glioma cells, (iii) adenosine A1 receptor-Gα1 inChinese hamster ovary cells, (iv) GABA_(B) receptors-Gα1 in rathippocampal CA1 pyramidal neurons, (v) muscarinic M2 receptor-Gα1 inhuman embryonic kidney cells, and the like. Any GPCR or group of GPCRwhich is convenient or desired can be used to define the interactionrequirements, and skilled workers are aware of many methods tounderstand structure-activity relationships in receptor binding of thiskind. Any of these methods are contemplated for use in these methods andmay substitute for the particular methods of the exemplified embodiment.

The plasmid display method provides an efficient means of identifyingspecific and potent peptides that can serve as competitive inhibitors ofprotein-protein interactions. Using the information gleaned fromstructure-activity studies, a library of variant peptides encodingsequences related to a GPCR-binding region, for example the Gα subunitcarboxyl terminus, for each of the classes of the Gα subtypes or Gβγ canbe prepared. Exemplary native sequences upon which libraries may bebased include those listed in Table III, below. Libraries advantageouslycontain peptides with computer-generated random substitutions within thesequence, and allow one to test a large number of peptide sequences atone time. Preferably, peptide sequences in each library are constructedsuch that approximately 50% of the amino acid residues are identical tothe native GPCR binding region and the remaining amino acid residues arerandomly selected from any amino acid. The peptides may range in sizefrom about 7 to about 55 amino acid residues or from about 8 to about 50amino acids long or from about 7 to about 70 amino acid residues orlonger, preferably from about 9 to about 23 amino acid residues or about9 to about 15 amino acid residues. Undecamer peptides are mostpreferred. Libraries may be constructed in which about 10% to about 90%of the amino acid residues unchanged from the native sequence; however,about 30% to about 70% unchanged is preferred and about 50% is mostpreferred.

Alternatively, a synthetic peptide library can be based on any proteinknown to interact with a GPCR, using randomly created overlappingregions of the protein. The peptides may be about 7-70 amino acids longor about 8-50 amino acids long or preferably about 9 to about 23 orabout 9 to about 15 amino acids long and most preferably about 11 aminoacids long. Oligonucleotides encoding the peptides advantageously may becloned to the 3′ end of the LacI gene, with a linker sequence at theN-terminus of the peptide. The linker sequence is not mandatory forsuccessful screening, but is generally preferred. Restriction enzymesites may be placed at either end of the peptide coding sequence forcloning purposes. See Table I below for a schematic representation of apeptide library and an example of one peptide. Additional peptides whichalso can be used are shown in Tables II and III, below. Theoligonucleotides encoding the actual peptide sequences are synthesizedwith 70% of the correct base and 10% each of the remaining bases,leading to a biased peptide library with an approximately 50% chance ofhaving the correct amino acid at any specific position along the peptidesequence. Different ratios of bases may be used to achieve the desiredmutagenesis rate at any particular position in the sequence. TABLE IExample for Construction of a Synthetic Peptide Library. (SEQ ID NO:13)                       Q  R  M  H  L  R  Q  Y  E  L  L gaggtggtnnknnknnknnk attcgtgaaaacttaaaagattgtggtcgtttc taa ctaagtaaagc   A           B                      C                  D       E(SEQ ID NO:14) n = any nucleotide base; k = guanidine or thymidine; A= restriction enzyme site; B = linker sequence; C = oligonucleotideencoding peptide sequence; D = stop codon; E = restriction enzyme site.

TABLE II Gα Subunit Peptides and Corresponding DNA Constructs. Gα SEQSubunit Sequence ID NO: Gt I K E N L K D C G L F 15 atc aag gag aac ctgaaa gac tgc ggc ctc ttc 16 Gi1/2 I K N N L K D C G L F 17 ata aaa aataat cta aaa gat tgt ggt ctc ttc 18 GRi1/2 N G I K C L F N D K L 19 aacggc atc aag tgc ctc ttc aac gac aag ctg 20 Gi3 I K N N L K E C G L Y 21att aaa aac aac tta aag gaa tgt gga ctt tat 22 Go2 I A K N L R G C G L Y23 atc gcc aaa aac ctg cgg ggc tgt gga ctc tac 24 Go1 I A N N L R G C GL Y 25 att gcc aac aac ctc cgg ggc tgc ggc ttg tac 26 Gz I Q N N L K Y IG L C 27 ata cag aac aat ctc aag tac att ggc ctt tgc 28 G11 L Q L N L KE Y N L V 2 ctg cag ctg aac ctc aag gag tac aac ctg gtc 29 Gq L Q L N LK E Y N A V 30 ctc cag ttg aac ctg aag gag tac aat gca gtc 31 Golf Q R NH L K Q Y E L L 32 cag cgg atg cac ctc aag cag tat gag ctc ttg 33 G14 LQ L N L R E F N L V 34 cta cag cta aac cta agg gaa ttc aac ctt gtc 35G15/16 L A R Y L D E I N L L 36 ctc gcc cgc tac ctg gac gag atc aac ctgctg 37 G12 L Q E N L K D I M L Q 38 ctg cag gag aac ctg aag gac atc atgctg cag 39 G13 L N D N L K Q L M L Q 40 ctg cat gac aac ctc aag cag cttatg cta cag 41 Gs Q R N H L R Q Y E L L 13 cag cgc atg cac ctt cgt cagtac gag ctg ctc 42 5′- gatccgccgccaccatggga- -tgaa-3′(SEQ ID NOS:43, 44)

TABLE III Exemplary Native C Protein Sequences for Library/MinigeneConstruction.* SEQ SEQ ID ID Name Sequence NO: Name Sequence NO: hGtIKENLKDCGLF 15 CryptoGbal LQNALRDSGIL 62 hGi1/2 IKNNLKDCGLF 17 GA3_USTLTNALKDSGIL 63 G05_DRO IKNNLKQIGLF 45 GA1_KLU IQONLKKSGIL 64 GAF_DROLSENVSSMGLF 46 GA3_UST LTNALKDSGIL 63 Gi-DRO IKNNLKQIGLF 45 GA1_DICNLTLGEAGMIL 64 hGi3 IKNNLKECGLY 21 GA2_KLU LENSLKDSGVL 65 hGO-1IANNLRGCGLY 25 GA2_UST ILTNNLRDIVL 66 hGO-2 IAKNLRGCGLY 47 Mgs-XLQRMHLPQYELL 67 GAK_CAV IKNNLKECGLY 21 hGs QRMHLRQYELL 13 GO_XENIAYNLRGCGLY 48 hGolf QRMHLKGYELL 68 GA3_CAEEL IQANLQGCGLY 49 GA1_COPCOLQLHLRECGLL 69 GA2_CAEEL IQSNLHKSGLY 50 GA1-SOL RRRNLFEAGLL 70 GA1_CAEELLSTKLKGCGLY 51 GA2_SB RRRNLLEAGLL 71 GAK_XEN IKSNLMECGLY 52 GA1_SBRRRNPLEAGLL 72 GA1_CAN VQQNLKKSGIM 53 GA1_UST IQVNLRDCGLL 73 hGZIQNNLKYIGLC 27 GA4_UST RENLKLTGLVG 74 hG15 LARYLDEINLL 36 GA1_ORYSADESMRRSREGT 75 GA2_SCHPO LQHSLKEAGMF 54 GQ1_DROME MQNALKEFNLG 76 hG12LQENLKDIMLQ 38 GA2_DIC TQCVMKAGLYS 77 hG13 LHDNLKQLMLQ 40 GS-SCHLQHSLKEAGMF 54 GAL_DRO LQRNLNALMLQ 55 GA-SAC ENTLKDSGVLQ 56 GA2_YSTENTLKDSGVLQ 56 GA1-CE IISASLKMVGV 78 hG14 LQLNLREFNLV 34 GA2-CENENLRSAGLHE 79 hG11 LQLNLKEYNLV 2 GA3-CE RLIRYANNIPV 80 hGQ LQLNLKEYNAV30 GA4-CE LSTKLKGGGLY 51 GQ_DROME LQSNLKEYNLV 57 GA5-CE IAKNLKSMGLC 81G11_XEN LQHNLKEYNLV 58 GA6-CE IGRNLRGTGME 82 Gq_SPOSC IQENLRLCGLI 59GA7-CE IQHTMQKVGIQ 83 GA1_YST IQQNLKKIGII 60 GA8-CE IQKNLQKAGMM 84GA1_NEUCR IIQRNLKQLIL 61 GAS-DIC LKNIFNTIINY 85*For production of minigene constructs each nucleotide sequence shouldbe constructed to encode the amino acids MG at the N-terminus of thepeptide by using 5′-gatccgccgccaccatggga-(SEQ ID NO:43) and-tgaa-3′ (SEQ ID NO:44).

The peptides advantageously are synthesized in a display system forconvenience and efficiency of performing the binding reactions. Forexample, plasmid or phage display systems, as are known in the art, maybe employed. While peptide display systems are preferred, any methodwhich allows efficient contact of the peptides with a GPCR anddetermination of binding may be used.

A peptide display (“peptides on plasmids”) library is a convenientsystem for use with this invention which exploits the high affinity bondbetween LacI and lacO. The “peptides on plasmids” display is preferredfor use with this invention for two major reasons. The technique iseasily set up in the laboratory. In addition, the fusion of the peptideat the carboxyl terminus of the presenting protein mimics the normalpresentation for carboxyl terminal peptides during the screen. If aminoterminal or interior peptides are being tested, the peptide may becloned at the appropriate position to mimic native presentation.

The “peptides on plasmids” method for testing carboxyl terminal peptidesgenerally works as follows. Persons of skill in the art will be able tomodify these methods as needed to accommodate different conditions usingthis general description and the examples below as a guide. A library ofpeptides is created by degenerate PCR based on the native GPCR-bindingpeptide of interest and fused to the carboxyl terminus of LacI. Thepeptide library is expressed via a plasmid vector carrying the fusiongene. The plasmid also contains the Lac operon (LacO), and when E. colitranscribes and translates the Lacl fusion protein, it binds back as atetramer to the encoding plasmid through its lacO DNA binding sequence,displaying the inserted sequences of interest on the plasmid. Followingtranscription and translation, variant peptides encoding differentsequences related to the native peptide sequence therefore are displayedas carboxyl terminal extensions of the lacl gene. Thus, a stableLacI-peptide-plasmid complex is formed which can be screened for bindingto receptor. Methods described in Gates et al., J. Mol. Biol.255:373-386, 1996, the disclosures of which are hereby incorporated byreference, are suitable. See Examples 7 and 9 for exemplary methods.

The E. coli strain used to display the peptides was ARI814, which hasthe following genotype: Δ(srl-recA) endA1 nupG lon-11 sulA1 hsdR17 Δ(ompT-fepC)266 ΔclpA319::kan ΔlacI lacZU118. The strain contains thehsdR17 allele that prevents restriction of unmodified DNA introduced bytransformation or transduction. The ompT-fepC deletion removes the geneencoding the OmpT protease, which digests peptides between paired basicresidues. The lon-11 and clpA mutations also limit proteolysis byATP-dependent, cytoplasmic proteases. The deletion of the lacI geneprevents expression of the wild-type lac repressor, which would competewith the fusion constructs for binding to the lacO sites on the plasmid.The lacZ mutation prevents waste of the cell's metabolic resources tomake β-galactosidase in the absence of the repressor. The endA1 mutationeliminates a nuclease that has deleterious effects on affinitypurification and the recA deletion prevents multimerization of plasmidsthrough RecA-catalyzed homologous recombination. This strain wasselected also for its robust growth properties and high yields ofimmunocompetent cells. Transformation efficiencies of 2×10¹⁰ coloniesper mg DNA typically were achieved. Although this strain of E. coli ispreferred, those of skill in the art are aware of many alternativeswhich are convenient for use with the methods described. Therefore, anysuitable and convenient bacterial strain known in the art iscontemplated for use with this invention.

The LacI-peptide fusion protein library may be released from thebacteria by gentle enzymatic digestion of the cell wall using lysozyme.After pelleting the cell debris, the lysate then can be added directlyto immobilized receptor for affinity purification or used withoutpurification. The display library of these peptides is screened toidentify those peptides which bind with high affinity to a particularGPCR. In this way, it is possible to screen for and identify highaffinity peptides which bind GPCR and can interfere with or enhanceactivation of the pre-selected specific G protein. The library can bescreened against any desired GPCR. Since the combinatorial librarycontains peptides based on a particular Gα or Gβγ subunit, any GPCRwhich binds to or mediates signaling through that subunit or class ofsubunits can be used. Multiple libraries, based on the carboxyl terminalsequences or other regions of different G protein subunits may beconstructed for screening the same or different GPCR.

To screen the plasmid display library, a G protein coupled receptor ofinterest advantageously may be immobilized on microtiter plates forscreening by ELISA. A plasmid preparation (bacterial lysate) then may beadded to the wells. This screening procedure, involving allowing thepeptides displayed on the library plasmids to bind receptor, issometimes referred to as “panning.” Sequences that bind the receptorstick to the well so that non-binding sequences can be removed by awashing step. The adherent plasmids then can be expanded and used totransform E. coli. The “panning” process generally is repeated 2 to 8times. In general, however, 3 to 4 sequential screens are sufficient andpreferred. In the later rounds of panning, parent peptide (wild typesequence) preferably is co-incubated with the plasmid preparation tobind receptors and serve as a competitive inhibitor. In this way, onlyhigh affinity sequences on the display library are captured by theimmobilized receptor. The same competitive inhibition advantageously maybe performed using a high affinity peptide or small molecule which hasalready been identified, rather than the native peptide. See FIG. 1 fora schematic diagram generally describing the “panning” procedure andExample 7 for a specific embodiment. The selection process in thisembodiment preferably is carried out in low salt buffers because highsalt concentrations destabilize the Lacl-lacO complex, and could lead topeptides becoming associated with the incorrect plasmid. For the samereason, the panning buffers preferably contain lactose, which causes theLacl to bind more tightly to lacO.

The selection process of this invention allows the identification ofpeptide sequences with higher and higher affinity binding with eachround of panning. For example, diversity in an unpanned library may looklike the sequences given in Table IV, below, i.e. highly randomized.After successive rounds of selection, the selected adherent peptideswould look more like those given in Table V, below. TABLE IV Diversityin Unpanned Gq Library. SEQ. ID NO. Native LQLNLKEYNLV 2 clone #1LLLQLVEHTLV 86 clone #2 HRLNLLEYCLV 87 clone #3 EQWNMNTFHMI 88 clone #4SQVKLQKGHLV 89 clone #5 LRLLL*EYNLG 90 clone #6 RRLKVNEYKLL 91 clone #7LQLRLREHNLV 92 clone #8 HVLNSKEYNQV 93

TABLE V Selection in Panned Gαll Library. SEQ ID NO. Native LQLNLKEYNLV2 Round 1 1 MKLNVSESNLV 94 2 LQTNQKEYDMD 95 3 LQLNPREDKLW 96 4RHLDLNACNMG 97 5 LR*NDIEALLV 98 6 LVQDRQESILV 99 Round 2 1 LQLKHKENNLM100 2 LQVNLEEYHLV 101 3 LQFNLNDCNLV 102 4 MKLKLKEDNLV 103 5 HQLDLLEYNLG104 6 LRLDFSEKQLV 105 Round 3 1 LQKNLKEYNMV 106 2 LQYNLMEDYLN 107 3LQMYLRGYNLV 108 4 LPLNPKEYSLV 109 5 MNLTLKECNLV 110 6 LQQSLIEYNLL 111

LacI is normally a tetramer and the minimum functional DNA bindingspecies is a dimer. Thus, the peptides are displayed multivalently onthe fusion protein, leading to binding to the immobilized receptor in acooperative fashion. This cooperative binding permits the detection ofbinding events of quite low intrinsic affinity. The sensitivity of theassay is an advantage in that initial hits of low affinity can beidentified, but the disadvantage is that the signal in the ELISA doesnot necessarily correlate with the intrinsic affinity of the boundpeptides.

One preferred ELISA, where signal strength is better correlated withaffinity, involves fusing the sequences of interest from a population ofclones in frame with a gene encoding a protein, for example E. colimaltose binding protein (MBP). Once the sequences have been transferredinto the monomeric fusion protein, they can be overexpressed in E. coliand used as either crude lysates or purified fusion proteins for assayby an ELISA which detects the protein bound to receptor or anyconvenient assay. Controls having the vector alone which expresses TGGGlinker only fused to MBP, or having Gt:340-350K341R peptide fused to MBPmay be used, if desired. Frozen cell stocks preferably are kept in 25%glycerol at −80° C. The high affinity Gα peptides fused to MBPpreferably are analyzed by ELISA, where the resulting signal correlatesto the peptide's affinity for light-activated rhodopsin. The MBP-peptidefusions are expressed and purified over an amylose affinity column andused to measure the relative affinities of peptides of interest. Thosesamples with an absorbance of at least two standard deviations abovebackground may be considered to contain high affinity binding peptides.Any desired cut-off point may be used, however, depending on the assayparameters and the needs of the operator.

A suitable ELISA may be performed as follows, however those of skill inthe art will be able to modify the techniques for the conditions intheir assays. Serial dilutions of MBP-peptide fusion proteins are addedto 96-well plates with immobilized light-activated rhodopsin previouslyblocked with 0.1% BSA. After 1 hour at 37° C., the wells are washed withphosphate buffered saline (PBS)/0.l% Tween 20, and probed with ananti-MBP antibody, followed by a goat-anti-rabbit antibody conjugated tohorseradish peroxidase. Color development of the assay is allowed toproceed for 20 minutes, after which the A₄₅₀ is measured on a microtiterplate reader. See Gilchrist et al., Methods Enzymol. 315:388-404, 2000,the disclosures of which are hereby incorporated by reference.

The purified fusion proteins can be further tested by measuring theirability to compete for the site of binding on the receptor using nativepeptide, a LacI-peptide fusion protein, or heterotrimeric G protein. Useof competitive ELISA allows one to calculate IC₅₀ values for the bindingof individual fusion protein to the immobilized receptor.

Peptide fusion proteins can be analyzed in a competitive ELISA formatusing a fusion protein co-incubation to prevent the binding of loweraffinity peptide fusion proteins to the GPCR. Any convenient proteinwhich does not interfere with peptide binding may be used, including forexample, glutathione-S-transferase, green fluorescent protein orubiquitin, however a maltose binding protein fusion protein such asMB-Gα_(t)340-35OK341R is preferred. Competitive ELISA indicates whichpeptide sequences have the highest affinity for light activatedrhodopsin. Several different assay formats are suitable. For example,synthetic Gt:340-350K341R peptide may be used to compete with the MBPfusion proteins containing the Gα high affinity peptides for binding. Inaddition, MBP fusion proteins containing the Gα high affinity peptidesmay be used to compete with LacI-Gt:340-350K341R peptide fusion proteinfor binding to light-activated rhodopsin. Recombinant heterotrimeric Gtalso may be tested against the high affinity peptides. The relativeaffinity of the variant peptides may be assessed using an ELISA formatwhere a constant concentration of MBP-Gα peptide fusion proteins iscompeted by serial dilutions of native peptide, LacI-Gαt peptide fusionprotein or recombinant heterotrimeric Gt. The wells advantageously maybe probed with an anti-MBP antibody to measure the amount of MBP-Gαpeptide fusion protein remaining bound. The dose-response curves may beanalyzed by non-linear regression to calculate an EC₅₀.

Cloning the library into pJS142 creates a BspEI restriction site nearthe beginning of the random coding region of the library. Conveniently,digestion with BspEI and ScaI allows the purification of a 900 base pairDNA fragment that may be subcloned into pELM3, a vector that directs theMBP fusion protein to the cytoplasm, a reducing environment.Alternatively, the fragment can be cloned into pELM15, a vector whichdirects the MBP fusion protein to the periplasm, an oxidizingenvironment. pELM3 and pELM15 are simple modifications of the pMALc2 andpMALp2 vectors, respectively, available commercially (New EnglandBiolabs). Digestion of pELM3 with AgeI and ScaI allows efficient cloningof the BspEI-ScaI fragment from the pJS142 library. Any suitable methodmay be used which is convenient to achieve the desired result.Modifications of these methods are well known by those of skill in theart of molecular biology and are contemplated for use here.

Proof that the high affinity peptides competitively bind to GPCR andinterfere with or enhance its recognition of G protein can be obtainedusing a competitive binding assay in the presence of a heterotrimeric Gprotein. For example, if rhodopsin is the GPCR used in the screen,heterotrimeric G protein, transducin (Gt) may be used. Gt bindsrhodopsin with multiple epitopes and is membrane-bound viamyristoylation of the α subunit and farnesylation of the γ subunitcarboxyl terminus. Poor binding by carboxyl terminal native peptide LacIconstructs and/or heterotrimeric Gt indicates high affinity binding ofthe MBP-peptide fusion proteins. An analogous strategy of panning,peptide synthesis and binding studies may be employed for determininghigh affinity peptides that bind any GPCR, for example the thrombinreceptors (PAR1, PAR3, PAR4), dopamine receptors (D1, D2, D3, D4, D5),vasopressin receptors (V1a, V1b, V2) and histamine receptors (H1, H2,H3), using carboxyl terminal peptide libraries for any Gα subunit, forexample Gαi, Gαs and Gαq. Once peptide analogs with higher bindingaffinities have been elucidated, they can be exploited to inhibit GPCR-Gprotein interaction.

The peptides selected by this method, characterized by high affinity,specific blockade of or enhancement of a desired GPCR-mediated signalingevent, may be used as therapeutic agents such as traditionalpharmaceuticals or gene therapies to treat disorders which would benefitby modifying GPCR activity or used to screen additional libraries ofcompounds able to compete with the high affinity peptide analogs or tomodulate (i.e., increase or decrease) the binding affinity of the highaffinity peptide analogs or the high affinity peptide analog-fusionproteins.

Any method known in the art for selecting and synthesizing smallmolecule libraries for screening is contemplated for use in thisinvention. Small molecules to be screened are advantageously collectedin the form of a combinatorial library. For example, libraries ofdrug-like small molecules, such as β-turn mimetic libraries and thelike, may be purchased from for example ChemDiv, Pharmacopia orCombichem or synthesized and are described in Tietze and Lieb, Curr.Opin. Chem. Biol. 2:363-371, 1998; Carrell et al., Chem Biol. 2:171-183,1995; U.S. Pat. Nos. 5,880,972, 6,087,186 and 6,184,223, the disclosuresof which are hereby incorporated by reference.

Any of these libraries known in the art are suitable for screening, asare random libraries or individual compounds. In general, hydrophiliccompounds are preferred because they are more easily soluble, moreeasily synthesized, and more easily compounded. Compounds having anaverage molecular weight of about 500 often are most useful, however,compounds outside this range, or even far outside this range also may beused. Generally, compounds having c logP scores of about 5.0 arepreferred, however the methods are useful with all types of compounds.Simple filters like Lipinski's “rule of five” have predictive value andmay be used to improve the quality of leads discovered by this inventivestrategy by using only those small molecules which are bioavailable. SeeLipinski et al., Adv. Drug Delivery Rev. 23:3-25, 1997.

Combinatorial chemistry small molecule “libraries” can be screenedagainst drug targets. The idea is that diversity of chemical structuresincreases the chances of finding the needle in the 10²⁰⁰ possible smallorganic molecule haystack. These collections provide an excellent sourceof novel, readily available leads. For example, ChemDiv uses more than800 individual chemical cores, a unique Building Block Library, andproprietary chemistry in designing its Diversity Collections (smallmolecule libraries) to assemble 80,000-100,000 compounds a year.CombiLab lead library sets of 200-400 compounds also can be produced asa follow-up. In addition, ChemDiv's compounds are designed to ensuretheir similarity to drugs adjusted according to proprietary algorithmsof “drug-likeness definitions” (group similarity and advanced neural netapproaches), and a variety of intelligent instruments for ADME&T(Absorption, Distribution, Metabolism, Excretion and Toxicity)properties prediction, such as partition coefficient, solubility,dissociation coefficients, and acute toxicity.

Thus, focused synthesis of new small molecule libraries can provide avariety of compounds structurally related to the initial lead compoundwhich may be screened to choose optimal structures. Preferably, alibrary of compounds is selected that are predicted to be “drug-like”based on properties such as pKa, log P, size, hydrogen bonding andpolarity. The inventive multi-step approach which yields high affinitypeptides in the first step, and small molecules in a subsequent stepreduces the number of artificial hits by eliminating the lower affinitysmall molecules that would be selected and have to be assayed in anormal high throughput screening method. In addition, it focuses thesearch for molecules that can modulate the binding of a peptide themimics the G protein rather than screening for binding to any site onthe receptor. Other advantages of this technology are that it is simpleto implement, amenable to many different classes of receptors, andcapable of rapidly screening very large libraries of compounds.

Screening of the peptides or small molecules may be performedconveniently using receptors from any source. Generally, it isconvenient to purify receptor from cells and reconstitute the receptorin lipid vesicles or to use membranes isolated from insect or mammaliancells that express or overexpress the receptor. PAR1 and rhodopsin areconvenient receptors, however any suitable receptor is contemplated foruse with this invention. The receptors used for screening may bepurified from a natural source or purified from cells which overexpressthe receptor and reconstituted in lipid vesicles. Membranes containingthe receptor may be prepared from cells which natively express thereceptor, for example Sf9 cells which express PAR1, or from cells whichhave been genetically engineered to express the receptor, for examplemammalian or insect cells overexpressing PAR1. Peptides identified fromscreening a receptor (PAR1) expressed by three different methods areshown in Tables XI, XII, and XIII. The results indicate the methods givesimilar results showing a high degree of conservation, (N348; L349)being identified for all three methods of receptor expression.Initially, it is advantageous to determine the binding affinity of thepeptide fusion protein or high affinity peptide against which thepeptides or small molecules are screened. This allows the amount ofreceptor and peptide MBP peptide fusion protein or small molecule in theassay to be optimized.

Generally, it is convenient to test the libraries using a one well-onecompound approach to identify compounds which compete with the peptidefusion protein or high affinity peptide for binding to the receptor. Asingle compound per well can be used, at about 1 μM each or at anyconvenient concentration depending on the affinity of the receptor forthe compounds and the peptide against which they are being tested.Compounds may be pooled for testing, however this approach requiresdeconvalution. Compounds may be pooled in groups of about 2 to about 100compounds per well, or more, or about 10 to about 50 compounds per wellat about 10 nM each or at any convenient concentration depending on theaffinity of the receptor for the compounds being tested. Severaldifferent concentrations may be used if desired. Peptides desirably arescreened using a pooled approach because of the layer members ofpeptides which are screened in the first instance. Peptides may bescreened individually as well, but preferably are screened in pools ofabout 10⁴-10¹² peptides per well or about 10⁸-10¹⁰ peptide per well ormost preferably about 10⁹ peptides per well.

ELISA, or any other convenient assay, such as fluorescence assays orradioimmunoassay may be used to determine (1) if one or more peptides ineach well reduce the amount of binding by the high affinity peptidefusion protein or high affinity peptide, or (2) if one or more peptidesin each well bind to the receptor. Compounds may be tested at a seriesof concentrations as well, and this generally is preferred if theaffinity of the peptide or peptide fusion protein is not known. In anELISA, wells in which the OD₄₅₀ is half or less than half than that ofcontrol wells (no tested compounds) generally are considered “positive”and may be further studied. Any suitable cut-off point may be used,however, depending on the assay components and the goals of the assay.

Screening against the high affinity peptide analogs can be performedusing the desired GPCR immobilized onto microtiter wells, biochips, orany convenient assay surface. Binding assays performed in solution alsoare suitable. One, several, or thousands of candidate small moleculepharmaceutical compounds can be screened for binding to the receptor inthe presence or absence of a high affinity peptide analog. The assayspreferably are performed in the presence of a high affinity bindingpeptide to ensure that only those candidate compounds which cansuccessfully compete for binding against the high-affinity bindingpeptide will be captured by the receptor. Alternatively, organiccompounds or small molecules which have been identified by screening ascompetitively binding in the presence of a high affinity peptide analogalso may be used as lead compounds in screening for further smallmolecule candidate compounds with even higher affinity. In eitherscreening process, binding may be detected by any convenient method, forexample by ELISA, fluorescence assays or radioimmunoassays.

By using a two-step protocol to identify compounds which block G proteinsignaling, high throughput screening of compounds and characterizationof the selected compounds is significantly reduced in both time andcost, because only potent and strongly binding compounds are selected.The first step of identification of high affinity peptides whichstrongly compete with G proteins for their site of binding on Gprotein-coupled receptors insures this because the high affinitypeptides are designed and tested for the particular desired bindingspecificity, ability to modify function within a cellular system andability to modify functions in vivo.

Preferably, only the most strongly binding and effective peptide analogsor small molecules are used in the second or subsequent screening step.This two- or multi-step protocol reduces the number of false positivesand identification of compounds which bind only weakly by eliminatingthe lower affinity small molecules that would be detected and thenfurther studied if a conventional high throughput screening method wereused. This method, therefore, is simple to implement, inexpensive,composed of only a few components, amenable to many different classes ofreceptors, and capable of rapidly screening large libraries ofcompounds. This method enables efficient identification of new classesof small organic peptidomimetic molecules that function as inhibitors orenhancers of receptor action, for example, thrombin receptor modifiers,dopamine receptor modifiers, histamine receptor modifiers, orvasopressin receptor modifiers. These identified compounds can target asingle GPCR, a class of GPCR, or block or enhance a single G proteinpathway activated by GPCR.

Thorough evaluation of the selected compounds (either peptides or smallmolecules) for use as therapeutic agents may proceed according to anyknown method. Properties of the compounds, such as pK_(a), log P, size,hydrogen bonding and polarity are useful information. They may bereadily measured or calculated, for example from 2D connection tables,if not already known prior to identification by the inventive method asa useful compound. Association/dissociation rate constants may bedetermined by appropriate binding experiments. Parameters such asabsorption and toxicity also may be measured, as well as in vivoconfirmation of biological activity. The screen may be optimized forsmall molecules according to methods known in the art. Additionally, itis preferable to use a software system for presentation of data thatallows fast analysis of positives. See Example 36 and FIG. 2.

Pharmaceutical preparations are prepared by formulating the peptides orsmall molecules identified by the inventive screen according to methodswell known in the art, with any suitable pharmaceutical excipient orcombination of pharmaceutical excipients. Preparations may be made foradministration by any route, such as intravenous, intramuscular,subcutaneous, oral, rectal, vaginal, transdermal, transmucosal,sublingual and the like, however, parenteral routes generally arepreferred for peptide preparations. Any suitable vehicle may be used,for example saline or lactated Ringer's, for intravenous administration.

Dosages for treatment of GPCR-related diseases or conditions will dependon many factors such as the nature of the disorder, the GPCR involved,the route of administration, factors relating to the general physicalcondition and health of the patient and the judgment of the treatingphysician. Persons of skill in the art are well aware of these factorsand consider manipulation of dosage to obtain an optimum result to beroutine. Generally, dosages for intravenous administration may varybetween about 0.01 mg/kg and 1000 mg/kg, however, this range can beexpanded depending on the patient's needs. Such an expanded range isconsidered within the scope of this invention.

Alternatively, peptides according to this invention may be provided tocells, in vivo or ex vivo, by delivery of an expression construct. Genetherapy can be performed in vivo as a direct introduction of the geneticmaterial. The in vivo gene transfer would introduce the oligonucleotidesencoding the peptides to cells at the site they are found in the body,for example to skin cells on an arm, or to lung epithelial cellsfollowing inhalation of the gene transfer vector. Alternatively, ex vivogene transfer, the transfer of genes into viable cells that have beentemporarily removed from the patient and are then returned followingtreatment (e.g. bone marrow cells) could also be employed.

Gene transfer vectors can be engineered to enter specific tissues orcells. Transductional targeting allows the gene transfer vectors tointeract with specific cell surface receptors. Transductional targetingalso can take advantage of the rate of cellular division by using genetransfer vectors that target rapidly dividing cells such as tumor cells.Transcriptional targeting recruits distinct cellular promoter andenhancer elements to influence transcription of the therapeutic gene.Transfection efficiencies are also enhanced by engineering vectors withmonoclonal antibodies, carbohydrate ligands, and protein ligands thathelp deliver genes to specific cells.

The gene transfer vectors used to produce the high affinity peptidesinside cells could be viral vectors (e.g. Retrovirus, Adenovirus,Adeno-Associated Virus, Herpes Simplex Virus, or Vaccinia Virus). As analternative, non-viral vectors also may be used, these include suchmethods as injection of naked DNA, or introduction of either DNA orpeptides by attachment to positively charged lipids, or cationicliposomes, electroporation or ballistic DNA injection (limited toex-vivo applications), as well as introduction of branched peptides.

Tet-inducible retroviral vectors for the native C-terminal sequencesthat co-expresses GFP driven by an internal ribosomal entry site (IRES)from encephalomyocarditis virus (p-Tet-Ti-GFP) may be used. Thesevectors can be modified so that they encode the high affinity peptidesequences. In addition, the high affinity peptide can be driven by asequence allowing for spatial or temporal expression. For in vitrostudies, viral supernatants may be collected from a pantropic producerline such as GP-293 (Clontech) in serum-free media. Viral supernatantsmay be concentrated by ultracentrifugation at 4° C. for 2 hr at 22,000rpm, and the pellets resuspended in 1/100 the original volume inserum-free media with a titer of at least 10⁸ IU (infectious units)/mland stored at −80° C.

Murine leukemia virus (MLV) derived retroviral vectors are commonly usedvehicles for stable delivery of therapeutic genes into endothelialcells. For the retrovirus studies in vivo, high affinity peptidesadvantageously are subcloned into a replication-defective murine Moloneyretrovirus vector which is Tet-inducible and co-expresses GFP driven byan internal ribosomal entry site (IRES) from encephalomyocarditis virus(pTet-GFP). These constructs may then be transiently transfected into aproducer line to generate cell-free titers of 10⁶-10¹⁰ IU/ml. If needed,a pantropic retroviral expression system which utilizes VSV-G, anenvelope glycoprotein from the vesicular stomatitis virus (GP-293;Clontech), may be utilized to overcome low transfection efficiencies. Byusing this innovative cell-based gene transfer method one can obtainstable, long-term, and localized gene expression of the high affinityC-terminal peptides.

To conclusively demonstrate that the compounds identified by this methodcan modulate G protein signaling events implicated in disease syndromesin vivo, antagonism or enhancement of selective G protein signaltransduction events may be confirmed. One method of testing the abilityof compounds to compete with native G protein binding involvesexpressing peptides that block the receptor-G protein interface in cellsbearing the receptor. Plasmid constructs that encode GPCR-binding regionpeptides, such as carboxyl terminal peptide sequences from the variousGα subunits (see Table VI) can be used to express them in cells in vivo,ex vivo or in vitro, so that the metabolic effects of selective GPCRblockade can be studied qualitatively and quantitatively. Such studiesprovide proof that the binding which the compounds possess is useful invivo to modulate selective G protein signals.

Expression of the peptides is conveniently achieved using the minigeneapproach by methods such as those described in Examples 23 and 24,however any suitable method may be used. Minigene vectors allow the highaffinity peptides to be evaluated in cellular systems prior to highthroughput screening. Any desired peptide sequence may be expressedusing these methods. Those of skill in the art are well aware ofalternative methods for construction, transfection and expression ofprotein and peptide constructs comprising the high affinity peptideanalogs, and such methods are contemplated for use with them.

Human embryonic kidney cells advantageously are cultured in DMEM (Gibco)with 10% fetal bovine serum (Gibco), and Pen/Strep (5000 U/ml; 5000pg/ml/ Gibco) in an atmosphere of 95% air/5% CO₂ at 37° C. The cells maybe plated at 60-70% density the day before transfection and transientlytransfected for 1.5 hours with DNA (3 μg) for pcDNA 3.1 vector with theinsert (pcDNA3.1-high affinity peptide) or vector alone using anEffectene kit from Qiagen. After transfection, cells are washed oncebefore adding complete HMEC media. When required, selection for cellscarrying the minigenes may be performed by adding Neomycin to the media48 hrs after transfection. To monitor efficiency of transfection cellsare transfected with the GFP plasmid (Clonetech). When necessary,transfectants may be selected using 300 μg/mL geneticin (G418). Theexpression of the vectors in HEK transfectants can be confirmed usingreverse transcription (RT)PCR and Northern blot analysis for mRNAexpression, and expression of the peptides can be characterized by HPLCas described previously. See Gilchrist et al., Methods Enzymol.344:58-69, 2002, the disclosures of which are hereby incorporated byreference. TABLE VI Exemplary Sequences of C-terminal Minigene Peptides.Peptide Name Sequence SEQ ID NO: Gαi MGIKNNLKDCGLF 112 GαiRMGNGIKCLFNDKL 113 Gαq MGLQLNLKEYNAV 114 Gαq** MGLQLNLKEYNTL 115 Gα12MGLQENLKDIMLQ 116 Gα13 MGLHDNLKQLMLQ 117

As discussed above, many receptors interact with and activate multiple Gproteins. Using the minigene strategy to introduce the highaffinity-binding carboxyl terminal peptides into cells, it is possibleto inhibit specific G protein-coupled receptor interactions withindividual G proteins, thus demonstrating the feasibility of specific Gprotein blockade in vivo with compounds identified by the inventivemethod. For those receptors which activate multiple G proteins each ofwhich activates a distinct set of signaling pathways mediating aspecific set of responses (for example, the thrombin receptor), onepathway can be inhibited without substantially affecting the others.

To selectively antagonize G protein signal transduction events in vivoby expressing peptides that block the receptor-G protein interface,minigene plasmid vectors were designed to express the C-terminal peptidesequence of the various Gα subunits following their transfection intomammalian cells. A control minigene vector also was created, encodingthe carboxyl terminus of Gαi_(1/2) in random order (GαiR, see Table VI).One important element necessary for the minigene approach to blockintracellular signaling pathways effectively in vivo is expression ofadequate amounts of the desired peptides. Therefore, expression of theminigene should be confirmed by a convenient method of detecting mRNA,protein or both. Any convenient method known in the art can be used.

To determine the cellular efficacy of the minigene approach forexpressing GPCR binding peptides, and to show the specific inhibition ofone G protein pathway in response to a given receptor activation signalwithout affecting others, compounds advantageously may be assayed in asystem designed to exhibit a measurable cellular signaling endpoint. Oneexample of such a system is the thrombin receptor, PAR1, in endothelialcells. This receptor activates multiple G proteins. Several signalingendpoints, including transcription analysis of induced PAR1 geneexpression; biochemical analysis of effector molecules including [Ca²⁺],MAP kinase (“MAPK”) activity, adenylyl cyclase activity, and inositolphosphate accumulation; as well as functional assays such as cellproliferation and endothelial permeability are available to measurespecific activation or modulation of activation of different G proteinsby ligand binding at this receptor. Signaling activity may be measuredby any convenient method, including: measuring inositol phosphateaccumulation; measuring intracellular calcium concentration levels;measuring transendothelial electrical resistance; measuring stress fiberformation; measuring ligand binding (agonist, antagonist, inverseagonist, etc.); measuring receptor expression; measuring receptordesensitization; measuring kinase activity; measuring phosphataseactivity; measuring nuclear transcription factors; measuring cellmigration (chemotaxis); measuring superoxide formation; measuring nitricoxide formation; measuring cell degranulation; measuring GIRK activity;measuring actin polymerization; measuring vasoconstriction; measuringcell permeability; measuring apoptosis; measuring cell differentiation;measuring membrane association of a protein that translocates upon GPCRactivation, such as protein kinase C; measuring cytosolic accumulationof a protein that translocates upon GPCR activation, such as proteinkinase C; measuring cytosolic accumulation of a protein thattranslocates upon GPCR activation, such as src; and measuring nuclearassociation of a protein that translocates upon GPCR activation, such asRan. The functional effects of Gα C-terminal minigenes in the mechanismof thrombin-induced cell retraction, as measured by the change intransendothelial electrical resistance (TEER) also can be used tomeasure G protein inhibition.

For example, thrombin-mediated PAR1 gene induction was inhibited inhuman microvascular endothelial cells (HMEC) expressing the Gαi minigeneconstruct. Expression of the Gαq minigene construct, however, affectedthrombin-mediated inositol phosphate accumulation. Expression of Gαqalso specifically decreased both thrombin-induced intracellular[Ca⁺⁺]_(i) rise and thrombin-induced MAPK activity.

Thrombin activation of the Gαi mechanism in HMEC decreases cAMP levelsincreased in response to isoproterenol (which acts through Gαs). Assayfor cAMP level increases in response to isoproterenol alone may becompared to increases after thrombin pre-incubation in cells expressingGαi to show that expression of the GPCR binding peptide blocks Gαisignaling.

Recent work by Gohla et al., J. Biol. Chem. 274:17901-17907, 1999,elegantly demonstrated that thrombin receptors induce stress fiberaccumulation via Gα12 in an EGF receptor-independent manner. Stressfiber formation appears to be Rho dependent. Both G12 and G13 have beenimplicated in the Rho signaling pathway. Therefore, expression of Gα12and Gα13 GPCR-binding peptides in HMEC were used to determine whetherthese peptides could block the appearance of stress fibers in responseto thrombin.

The extracellular signal-regulated kinase (ERK) subfamily ofmitogen-activated protein kinases (MAPKs) regulates numerous cellsignaling events involved in proliferation and differentiation. Thisforms the basis of another assay which can determine whether GPCRbinding peptides can affect a specific G protein mediated pathway.Transfection of HMEC cells with minigenes encoding GPCR binding peptidesalong with HA-MAPK followed by immunoprecipitation of the HA-MAPKpermits measurement of the effects only on cells expressing GPCR bindingpeptides.

Many studies have shown that the M₂ muscarinic receptor (mAChR) couplesexclusively to the Gi/GO family. See Dell'Acqua et al., J. Biol. Chem.268:5676-5685, 1993; Lai et al., J. Pharm. Exp. Ther. 258:938-944, 1991;Offermanns et al., Mol. Pharm. 45:890-898, 1994; Thomas et al., J.Pharm. Exp. Ther. 271:1042-1050, 1994. The M₂ mAChR can efficientlycouple to mutant Gαq** in which the last five amino acids aresubstituted with the corresponding residues from Gαi or GαO, suggestingthat this receptor contains domains that are specifically recognized bythe carboxyl terminus of Gαi/O subunits. See Liu et al., Proc. Natl.Acad. Sci. USA 92:11642-11646, 1995.

To test inhibition of G protein-coupled receptor-mediated cellularresponses by carboxyl terminal Gα peptides expressed using minigeneconstructs, prototypical directly Gβγ activated channels (GIRK channels)regulated by a pertussis toxin-sensitive M₂ mAChR was chosen as themodel. In this model, the importance of the Gα carboxyl terminus and thedownstream effector system have been well established. See Krapivinskyet al., J. Biol. Chem. 270:29059-29062, 1995; Krapivinsky et al., J.Biol. Chem. 273:16946-16952, 1998; Sowell et al., Proc. Natl. Acad. Sci.USA 94:7921-7926, 1997. Inhibition of M₂ mAChR activation of inwardlyrectifying potassium currents can be tested to demonstrate inhibition ofa downstream functional response following agonist stimulation of GPCRon cells transiently transfected with a Gα carboxyl terminal peptideminigene or treated with a pharmaceutical compound identified byscreening against high affinity Gα peptides.

GIRK channels modulate electrical activity in many excitable cells. SeeBreitwiese et al., J. Membr. Biol. 152:1-11, 1996; Jan et al., Curr.Opin. Cell Biol. 9:155-160, 1997; Wickman et al., Curr. Opin. Neurobiol.5:278-285, 1995. Because the channel opens as a consequence of a directinteraction with Gβγ, whole cell patch clamp recording of I_(KACh) canbe used to demonstrate inhibition of a downstream functional responsefollowing agonist stimulation of GPCR on cells transiently transfectedwith a Gα carboxyl terminal peptide minigene or treated with apharmaceutical compound identified by screening against high affinity Gαpeptides. Superfusion of cells expressing GIRK1/GIRK4 with their ligand,acetylcholine (ACh), activates inwardly rectifying potassium currents.

Using well-established receptor models accepted to be indicative of invivo cellular results, this type of data can show that the individual Gproteins activated via a given GPCR have specific roles in mediatingcellular events and can be modulated in a specific fashion by ligandsmimicking GPCR binding regions of individual Gα subunits. In particular,for receptors such as the thrombin receptor, which activate multiple Gproteins, each of which activates a distinct set of signaling pathwaysmediating a specific set of responses, it is possible using theinventive methods to block one pathway while leaving all the othersfunctional. The high affinity peptide analogs identified in vitro byconsecutive affinity purification and competitive binding are capable ofspecifically inhibiting the downstream consequences of G proteinsignaling.

The assays described above clearly establish the ability of compoundsidentified by in vitro competitive binding studies to modulate aparticular GPCR-G protein interaction selectively, even when the GPCRregulates multiple G proteins within the cell. Moreover, the peptidescompete very effectively with the native sequence. In addition, theminigene approach described above and exemplified in the examples belowallows a systematic test of the roles of other G proteins such as Gα12and Gα13, which may be involved in the mechanism of increase ofendothelial permeability, and clearly demonstrates the viability of thisapproach to select and identify Gα subunit modulating compounds. Thepeptides therefore are suitable for use in treatment of any disorder orsyndrome characterized by G protein signaling excess.

In another aspect, the invention relates to methods to identify the Gproteins with which a specific orphan receptor is coupled, using thematerials provided by the invention. For example, the described methodscan be used to test any GPCR with a battery of Gα subunit peptides todetermine which species of G protein(s) mediates the effects of thereceptor. The methods described in Examples 12-14 are suitable. Those ofskill in the art are capable of designing other assays, or variationsand modifications using these assays as guides.

Rhodopsin can be measured spectrophotometrically in many of itsconformational states. The high affinity, biologically active rhodopsinstate can be easily differentiated from its precursor, MI, by the“extra” MII assay. See Example 35. This assay relies on the observationthat under conditions of high pH and low temperature, MII is stabilizedin the presence of Gt and can be spectrophotometrically measured. Theability of the C-terminal peptide to stabilize Meta II in the samemanner as the heterotrimeric Gt, provides the tools to investigate thestructural basis of the interaction of G proteins with the agonistbinding sites of activated receptors.

The screening platform according to one embodiment of the invention canidentify small molecules that increase the binding of the high affinitypeptides which mimic G protein or stabilize the active conformation ofthe GPCR. These small molecules have an appropriate dose curve, and havean EC₅₀ in the low μM range. Samples of urea-washed rod outer segmentstypically have little or no (<5%) stabilization of MII unless G proteinis added, however, the small molecules identified by the invention inone example screen stabilize the active (signaling) conformation ofrhodopsin (MII). Addition of the small molecule alone results in 70%stabilization. The EC₅₀ for stabilization of MII also appears to be inthe low μM range. Further, these same small molecules were added tohuman embryonic kidney (HEK) cells with measurement of their calciumresponse to a second GPCR do not enhance signaling of an unrelated GPCRand do not appear to cause an acute toxic response.

Drug discovery has evolved from an essentially random screening ofproducts, into a process that includes the rational and combinatorialdesign of large numbers of synthetic molecules as potential bioactiveagents, such as agonists, antagonists and inverse agonists, as well asthe structural characterization of their biological targets, which maybe polypeptides, proteins, or nucleic acids. Several approaches tofacilitating the understanding of the structure of the therapeutictargets have been developed. These include sequencing of proteins andnucleic acids (Findlay et al., Protein Sequencing: A Practical Approach,IRL Press, Oxford, 1989; Adams et al., In Automated DNA sequencing andAnalysis, Academic Press, San Diego, 1994), elucidation of secondary andtertiary structures via NMR (Jefson, Ann. Rep. Med. Chem. 23:275, 1998;Erikson and Fesik, Ann. Rep. Med. Chem. 27:271-289, 1992), X-raycrystallography (Erikson and Fesik, Ann. Rep. Med. Chem. 27:271-289,1992) and computer algorithms for predicting protein folding (Copeland,Methods of Protein Analysis: A Practical Guide to Laboratory Protocols,Chapman and Hall, New York, 1994; Creighton, Protein Folding, W.H.Freeman and Co., 1992). Experiments such as ELISA (Kemeny andChallacombe, ELISA and other Solid Phase Immunoassays: Theoretical andPractical Aspects; Wiley, New York, 1988) and radioligand binding assays(Berson and Yalow, Clin. Chim. Acta, 22:51-60, 1968; Chard, AnIntroduction to Radioimmunoassay and Related Techniques, Elseveierpress, Amsterdam/New York, 1982), surface-plasmon resonance (Karlsson etal., Anal. Biochem. 300:132-138, 2002), and scintillation proximityassays (Kariv et al., J. Biomol. Screen. 4:27-32, 1999) also can be usedto understand the nature of the receptor-G protein interaction.

Peptides that block the protein-protein interactions of interest do soby binding to the surface of one of the interacting proteins andmimicking the interactions of the complete protein with the receptor.One can study the conformation of the active receptor-bound peptideswhen they are exchanging with the bound form, using transferred-NOESYNMR methods or X-ray diffraction if the peptides are more tightly bound.The bound peptide conformations can provide useful templates for thedesign of non-peptide small molecule drug leads which block theprotein-protein interactions of interest. The binding sites of thepeptides on the receptors also can be investigated using photochemicalcrosslinking, by substitution of peptide residues with photoactivatableamino acid analogs, crosslinking of the peptide to the receptor bindingsites, cleavage of the receptor into peptide fragments and massspectrometry analysis of the location of the binding sites. Combiningstructural data from a variety of experiments allows the development ofmodels of the interacting protein surfaces using computer graphics andguides the design of novel non-peptide molecules to modulate theinteractions.

In X-ray diffraction crystallography, a crystalline form of the moleculeunder study is exposed to a beam of X-rays and the intensity ofdetracted radiation is measured at a variety of angles from the angle ofincidence. The beam of X-rays is diffracted into a plurality ofdiffraction “reflections,” with each reflection representing areciprocal lattice vector. From the diffraction intensities of thereflections, the magnitudes of a series of numbers, known as “structurefactors,” are determined. The structure factors in general are complexnumbers, having a magnitude and a phase in the complex plane, and aredefined by the electron distribution within the unit cell of thecrystal.

Crystals can be formed of receptor or portions of receptor bound topeptides that stabilize a particular conformation of interest. Themethods of this invention, which identify peptides using combinatorialtechniques that scan the complete set of possible amino acid sequencesto find those that bind specifically to a particular receptor with highaffinity, can identify peptides that bind to particular conformations ofa GPCR. These peptides can be bound (and co-crystallized) with thereceptor for structural determination studies by NMR or crystallography.Co-crystallization in this manner may be performed according to anymethod known in the art, for example the methods of Kimple et al.,Nature 416:878-881, 2002, the disclosures of which are herebyincorporated by reference.

Therefore, in another embodiment, assays for identifying peptides thatbind to a particular conformer of a GPCR are performed according to themethods described above for selection of the high affinity peptideanalogs that bind activated rhodopsin. Once the high affinity peptideshave been identified, they can be used in peptidomimetic studies.Compounds that mimic the conformation and desirable features of aparticular peptide, e.g., an oligopeptide, but that avoid undesirablefeatures, e.g., flexibility (loss of conformation) and metabolicdegradation, are known as “peptidomimetics.” Peptidomimetics that havephysical conformations that mimic the three dimensional structure of thehigh affinity peptide analogs, that have surface active groups thatallow binding to the receptor, or that have physical conformations thatmimic the three dimensional structure of the high affinity peptideanalogs can be used to make pharmaceutical compositions. Drugs with theability to mimic the function of the high affinity peptide analogs thatbind to the designated receptors can be identified using rational drugdesign according to this invention. The compounds preferably include thesurface active functional groups of the high affinity peptide analogs,or substantially similar groups, in the same or substantially similarorientation, so that the compounds possess the same or similarbiological activity. The surface-active functional groups in the highaffinity peptide analogs possess a certain orientation when the receptoris present, in part due to their secondary or tertiary structure.Rational drug design involves both the identification and chemicalmodification of suitable compounds that mimic the function of the parentmolecules.

The physical conformation of the peptidomimetics are determined, inpart, by their primary, secondary and tertiary structure. The primarystructure of a peptide is defined by the number and precise sequence ofamino acids in the high affinity peptide analogs. The secondarystructure is defined by the extent to which the polypeptide chainspossess any helical or other stable structure. The tertiary structure isdefined by the tendency for the polypeptides to undergo extensivecoiling or folding to produce a complex, somewhat rigidthree-dimensional structure.

Computer modeling technology allows scientists to visualize thethree-dimensional atomic structure of a selected molecule and deriveinformation that allows the rational design of new compounds that willmimic the molecule or which will interact with the molecule. Thethree-dimensional structure can be determined based on data from X-raycrystallographic analyses and/or NMR imaging of the selected molecule,or from ab initio techniques based solely or in part on the primarystructure, as described, for example, in U.S. Pat. No. 5,612,895. Thecomputer graphics systems enable one to predict how a new compound willlink to the target molecule and allow experimental manipulation of thestructures of the compound and target molecule to perfect bindingspecificity.

Many databases and computer software programs are available for use indrug design. For example, see Ghoshal et al., Pol. J. Pharmacol.48(4):359-377, 1996; Wendoloski et al., Pharmacol. Ther. 60(2):169-183,1993; and Huang et al., J. Comput. Aided Mol. Des. 11:21-78, 1997.Databases including constrained, metabolically stable non-peptidemoeties may be used to search for and to suggest suitable analogs of thehigh affinity peptides identified in the screen. Searches can beperformed using a three dimensional database for non-peptide (organic)structures (e.g., non-peptide analogs, and/or dipeptide analogs) havingthree dimensional similarity to the known structure of the activeregions of these molecules. See, for example, Allen, Acta Crystallogr.B. 58:380-388, 2002.

Alternatively, three dimensional structures generated by other meanssuch as molecular mechanics can be consulted. In addition, searchalgorithms for three dimensional database comparisons are available inthe literature. Rufino et al., J. Comput. Aided Mol. Des. 8:5-27, 1994.Commercial software for such searches is also available from vendorssuch as Accelrys Inc. (9685 Scranton Road, San Diego, Calif.92121-3752). The searching is done in a systematic fashion by simulatingor synthesizing analogs having a substitute moiety at every residuelevel. Preferably, care is taken that replacement of portions of thebackbone does not disturb the tertiary structure and that the side chainsubstitutions are compatible to retain the high affinitypeptide/receptor interactions.

Using information regarding the bond angles and spatial geometry of thecritical amino acids, one can use computer programs as described hereinto develop peptidomimetics. Thermal protein unfolding, or thermal“shift” assays have been used to determine whether a given ligand bindsto a target receptor protein. In a physical thermal shift assay, achange in a biophysical parameter of a protein is monitored as afunction of increasing temperature. For example, in calorimetricstudies, the physical parameter measured is the change in heat capacityas a protein undergoes temperature-induced unfolding transitions.Differential scanning calorimetry may be used to measure the affinity ofa ligand for a G protein coupled receptor. Grauschopf et al.,Biochemistry 39:8878:87, 2000; Brandts et al., Biochemistry 29:6927-40,1990. Thus, using methods common to those skilled in the arts, the highaffinity peptides may be assayed for their ability to modulate thermalshift of the receptor.

Because of the difficulty in obtaining high-resolution crystallographicstructures from GPCRs, a variety of biophysical methods have beenapplied to characterize the interactions between the G protein, thereceptor and the ligand. These include fluorescence resonance energytransfer (FRET) experiments performed with fluorescence-labeled peptideanalogs (Bettio et al., Biopolymers 60:420-37, 2001), bioluminescenceresonance energy transfer (BRET) experiments (Ayoub et al., J. Biol.Chem. 277:21522-8, 2002), photoaffinity labeling (Turek et al., J. Biol.Chem 277:16791-16797, 2002), fluorescence spectroscopy (Ghanouni et al.J. Biol. Chem. 276:24433-24436, 2001), site-direct spin labeling(Hubbell et al., Nat. Struct. Biol. 7:735-739, 2000), Fourier transforminfrared difference spectroscopy (Vogel et al., Biochemistry35:11149-11159,1996), and intrinsic tryptophan fluorescencespectroscopy. Farrens et al., J. Biol. Chem. 270:5073-5076, 1995.

The following non-limiting examples are provided to illustrate certainaspects of this invention.

EXAMPLES Example 1 Construction of a Peptide Library

Construction of a biased peptide library has been described previously.Martin et al., J. Biol. Chem. 271:361-366, 1996; Schatz et al., Meth.Enzymol. 267:171-191, 1996. The vector used for library construction waspJS142 (see FIG. 3). This vector had a linker sequence between the LacIand the biased undecamer peptide coding sequence, as well as restrictionsites for cloning the library oligonucleotide. The oligonucleotidesynthesized to encode the mutagenesis library was synthesized with 70%of the correct base and 10% of each of the other bases at each position.This mutagenesis rate leads to a biased library such that there isapproximately a 50% chance that any of the 11 codons will be theappropriate (native) amino acid and approximately a 50% chance that itwill be another amino acid. In addition, a linker of four random NNK(where N denotes A, C, G or T and K denotes G or T) codons weresynthesized at the 5′ end of the sequence to make a total of 15randomized codons. Using this method, a library with greater than 10⁹independent clones per microgram of vector used in the ligation wasconstructed based on the carboxyl terminal sequence of Gαt (IKENLKDCGLF;SEQ ID NO:15). The nucleic acid used for creating this library was:5′-GAGGTGGTNNKNNKNNKNNKattcaaggagaacctgaaggactgcggcctcttcTAACTAAGTAAAGC-3′, wherein N=A/C/G/T and K=G/T; SEQ ID NO:118).

Example 2 Sequences for the Creation of Gα Subunit Peptide Libraries

Libraries were created using the methods of Example 1 and the sequenceslisted below in Table VII. TABLE VII C-Terminal Gα Subunit PeptideLibrary Constructs. Gα SEQ Sub- ID unit RE Linker Peptide Coding RegionStop RE NO: Gs 5-GAGGTGGT NNKNNKNNKNNK attcgtgaaaacttaaaagattgtggtcgtttcTAA CTAAGTAAAGC-3′ 14 G11 5-GAGGTGGT NNKNNKNNKNNKctgcagctgaacctgaaggagtacaatctggtc TAA CTAAGTAAAGC-3′ 119 G12 5-GAGGTGGTNNKNNKNNKNNK ctgcaggagaacctgaaggacatcatgctgcag TAA CTAAGTAAAGC-3′ 120G13 5-GAGGTGGT NNKNNKNNKNNK ctgcatgacaacctcaagcagcttatgctacag TAACTAAGTAAAGC-3′ 121 G15 5-GAGGTGGT NNKNNKNNKNNKctcgcccggtacctggacgagattaatctgctg TAA CTAAGTAAAGC-3′ 122 Gz 5-GAGGTGGTNNKNNKNNKNNK atacagaacaatctcaagtacattggcctttgc TAA CTAAGTAAAGC-3′ 123

Example 3 Isolation of Membranes from Insect Cells Expressing ThrombinReceptor

Sf9 cells (2×10⁸ cells) were cultured with 200 ml of Grace's insect cellculture medium (Life Technologies, Inc., Grand Island, N.Y.) containing0.1% Pluronic F-68 (Life Technologies, Inc., Grand Island, N.Y.)), 10%fetal calf serum, and 20 μg/ml gentamicin in a 1-liter spinner flask at27° C. for 25 hours. Sf9 cells were infected with the ThR/pBluebacrecombinant virus at a multiplicity of infection of 3-5, and cultured at27° C. for 4 days. The cells were harvested, washed with phosphatebuffered saline, and then resuspended in 10 mM Tris-HCl, pH 7.4. Cellswere then homogenized with a hand-held homogenizer set at low speed for20 seconds. The broken cells then were sedimented at 17,000×g for 15minutes. The supernatant was discarded, and the pellet resuspended in abuffer consisting of 50 mM Tris-HCl, pH 7.4 and 10% glycerol.Concentration of receptor in the membrane preparation ranged from1-10,000 pmole/mg. For screening, a final concentration of 200 μg/ml wasused. The thrombin receptors were tested for their ability to bind tothe native Gq-C terminal peptide using a MBP-Gq fusion protein.

Example 4 Isolation of Membranes from Mammalian Cells OverexpressingThrombin Receptor

PAR1 receptor cDNA (2.1 kb insert) was obtained by polymerase chainreaction and cloned into the mammalian expression vector pBJ5. Theresulting plasmid was transfected into Chinese hamster ovary cells bythe calcium phosphate coprecipitation method. The PAR1-transfected cellswere grown with Dulbecco's modified Eagle's medium containing 10% fetalcalf serum, 100 units/mL penicillin and 100 μg/mL streptomycin. Thecells were detached using PBS with 5 mM EDTA and washed twice in PBS.The pellet was either used immediately for membrane preparation orstored frozen at −20° C. Pellets were homogenized in 20 mM Tris-HCl, pH7.5, with 5 mM EDTA and 0.5 mM PMSF, using a Dounce homogenizer (10strokes) and sonicated for 10 seconds. Nuclear debris and intact cellswere removed by centrifugation at 3000 rpm for 10 minutes. Thesupernatant was sedimented at 12,000×g for 30 minutes and the resultingpellet suspended in 25 mM Tris-HCl, pH 7.5, 25 mM MgCl₂, 10% sucrose,0.5 mM PMSF, 50 μg/mL antipain, 1 μg/mL aprotinin, 40 μg/mL bestatin,100 μg/mL chymostatin, 0.5 μg/mL leupeptin and 0.7 μg/mL pepstatin. Themembranes were aliquoted and frozen at −80° C.

Example 5 Preparation of Rod Outer Segments

Bovine rod outer segments (rhodopsin-containing membranes) were preparedfrom fresh retinas under dim red light as described by Arsharky et al.,J. Biol. Chem. 269:19882-19887, 1994. The retinas were placed in abeaker for dissection filled with 200 mL of 30% (w/v) sucrose inisolation buffer (90 mM KCl, 30 mM NaCl, 2 mM MgCl₂, 0.1 mM EDTA, 1 mMDTT, 50 μM phenylmethylsulfonyl fluoride, 10 mM MOPS, pH 7.5) on icewith constant moderate stirring of the solution during dissection.Following dissection, the retina solution was left in the dark for onehour on ice. The retina-sucrose solution was distributed into eight 50mL tubes and sedimented at 3000×g for four minutes at 4° C. Thesupernatant was decanted into eight fresh centrifuge tubes and placed onice. The tubes were filled to 1.5 cm below top with isolation buffer,then sedimented at 17,000×g for 20 minutes (“spin 1”).

The pellets were resuspended in a small volume of 30% sucrose andconsolidated from eight tubes into four tubes. The tubes were filled to1.5 cm below top with 30% sucrose, sedimented at 5000×g for four minutesat 4° C., and the supernatant decanted into four clear tubes. Thesetubes were filled to 1.5 cm below top with isolation buffer andsedimented at 17,000×g for 20 minutes at 4° C. (“spin 2”).

A stepwise sucrose gradient was prepared in six gradient tubes using thesolutions in Table VIII, below, with a sequence from top to bottom of#2, #3, #4. TABLE VIII Sucrose Gradient Solutions. Solution #2 (0.84 M)#3 (1.0 M) #4 (1.14 M) 42% Sucrose 51.30 g 61.05 g 69.75 g 1.0 M MOPS750 μL 750 μL 750 μL 2.0 M KCl 2250 μL 2250 μL 2250 μL 3.0 M NaCl 750 μL750 μL 750 μL 2.0 M MgCl₂ 75 μL 75 μL 75 μL Total Weight 83.25 g 84.75 g86.25 g

The pellets from “spin 1” and “spin 2” were resuspended in isolationbuffer using 1 mL 26% sucrose buffer per tube. After making a slurry,each tube was homogenized with a 1 mL pipette and the tubesconsolidated. The pellet solution was carefully laid onto the sucrosegradients and was not allowed to invade the gradient layers. Thegradient tubes were subjected to 24,000×g for 30 minutes at 4° C. in aswinging bucket rotor, after which the orange layer containing themembranes was collected carefully, to avoid disturbing the pellet or thedark solution near the pellet. The membranes were distributed into six50 mL tubes and placed on ice. The tubes then were filled to 1.5 cmbelow top with isolation buffer and sedimented at 17,000×g for 20minutes at 4° C. The supernatant was discarded and the pelletsresuspended in 1 mL isolation buffer containing 5 μg/mL pepstatin and 10μg/mL E-64. This suspension was stored in a foil-wrapped 15 mL conicaltube at −80° C. until needed, then thawed, homogenized in EDTA buffer(10 mM Tris, pH 7.5, 1 mM EDTA 1 mM DTT) and sedimented at 30,000×g for30 minutes. The supernatants were discarded and the pellets resuspendedand sedimented again as described above. The pellets then wereresuspended in urea buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM DTT, 7 Murea), homogenized and sedimented at 45,000 kg for 40 minutes. Thesepellets were resuspended and homogenized in Buffer A (200 mM NaCl, 10 mMMOPS, pH 7.5, 2 mM MgCl₂, 1 mM DTT, 100 μM PMSF), then sedimented at30,000×g for 30 minutes. The pellets each were resuspended andhomogenized by pipetting in 1 mL buffer A and stored at −80° C. in 100μL aliquots in foil-covered tubes for use in assays. For screening, thereceptor was added to wells at 10 μg/ml. Binding assays were performedas in Example 18.

Example 6 Purification of PAR1 Thrombin Receptor from Insect Cells andReconstitution of Receptors into Lipid Vesicles

Sf9 cells (2×10⁸ cells) were cultured in Grace's insect cell culturemedium (Life Technologies, Inc., Grand Island, N.Y.) containing 0.1%Pluronic F-68 (Life Technologies), 10% fetal calf serum and 20 μg/mLgentamicin in a 1 L spinner flask at 27° C. for 25 hours. The cells wereinfected with ThR/pBluebac (recombinant virus) at a multiplicity ofinfection of 3-5 and cultured at 27° C. for four days. The cells wereharvested, washed with phosphate buffered saline containing 2.7 mM EDTAand stored at −70° C. until used. The cells were resuspended in lysisbuffer (2.5 mM Tris-HCl, pH 7.2, 7.5 mM NaCl, 10 mM EDTA, 1 mMphenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, 10 mg/mL aprotinin,50 mM NaF) and washed. All subsequent steps were done on ice with coldbuffers and centrifuge rotors at or below 4° C. The cells werehomogenized for one minute at maximum speed and sedimented for 45minutes at 30,000×g. The pellet was resuspended in lysis buffer and thehomogenation/washing step repeated three times. The resulting pellet wasresuspended in 30 mL solubilization buffer (20 mM Tris-HCl, pH 7.4, 15mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, 10mg/mL aprotinin, 50 mM NaF, 0.1% (w/v) digitonin, 0.1% (w/v) Nadeoxychoate) and then homogenized for one minute. The suspension wasstirred for 90 minutes at 4° C. and then sedimented for 60 minutes at30,000×g. The supernatant was loaded onto an anti-PAR1 monoclonalantibody column equilibrated with solubilization buffer containing 0.2%digitonin. After application of the supernatant, the column was washedwith 10 column volumes of 10 mM Tris-HCl buffer, pH 7.4, containing 0.2%(w/v) Na dodecyl maltoside. The receptor was eluted using 10 mMtriethylamine, pH 11.8. The eluted fractions were neutralizedimmediately using 1 M HEPES, pH 6.4. The pooled fractions were dialyzedagainst 50 mM HEPES buffer, pH 7.4, containing 50% (v/v) glycerol, 0.1 MNaCl and 0.2% (w/v) Na dodecyl maltoside. Aliquots were stored at −80°C.

For preparation of lipid vesicles, 200 μL phosphatidylserine (50 mg/mLin CHCl₃; Matreya) was dried in a rotary evaporator for 30 minutes orusing a stream of dry N₂. After addition of 200 μL buffer A (50 mMHEPES, 100 mM NaCl, 0.2% (w/v) Na dodecylmaltoside), the tube was sealedunder an N₂ atmosphere and sonicated in a bath sonicator for 30 minutes.Reconstitution of receptors into lipid vesicles was performed the sameday, using purified receptor prepared as in Example 5. Purified receptorstocks (200 μg/mL) were thawed on ice and 50 μL was incubated for 20minutes at 4° C. with the appropriate agonist peptide (100 nM finalconcentration). In the case of thrombin receptor, the agonist isthrombin receptor agonist peptide (CalbioChem). After addition of 80 μLsonicated lipids and 50 μL buffer A, the samples were mixed using avortex machine and placed on ice for 10 minutes. The samples then wereloaded onto a 1 mL Extracti-gel™ column which had been washed with 0.2%BSA and pre-equilibrated with 5 mL Buffer A without Na dodecylmaltoside.The reconstituted vesicles were eluted from the column with 2.5 mL HEKbuffer.

Samples (100-200 μL) were collected for purity analysis by SDS-PAGE. Theconcentration for each batch generally was about 10-1000 μg/ml. For use,receptor was placed in microtiter plates at about 1-100 μg/ml. Thepurified, reconstituted thrombin receptors were tested for their abilityto bind to the native Gq-C terminal peptide using a MBP-Gq fusionprotein. As a control, empty vesicles also were tested for their abilityto bind to the native Gq-C terminal peptide using a MBP-Gq fusionprotein.

Example 7 Identification of GPCR-Binding High Affinity Peptide Analogs(Panning)

Electrocompetent cells were produced as follows. A single colony ofARI814 bacteria was grown overnight at 37° C. in 5 ml sterile SOP (20g/L Bacto-tryptone; 10 g/L Bacto-yeast extract; 5 g/L NaCl; 2.5 g/Lanhydrous K₂HPO₄; 1 g/L Mg₂SO₄.7H₂O). One milliliter of this overnightgrowth was added to 500 ml SOP and the bacteria were allowed to growuntil the OD₆₀₀ read 0.6-0.8. All further washing steps were done in thecold. The cells were placed in an ice-water bath for at least 15minutes, then subjected to centrifugation at 4000×g for 15 minutes at 4°C. followed by resuspension in 500 ml 10% glycerol. After sitting on icefor 30 minutes, the cells were washed twice more in 500 ml and 20 ml 10%glycerol with sedimentation as above, and finally sedimented at 5000×gfor 10 minutes at 4° C. and resuspended in 1 mL 10% glycerol. Cells werequick-frozen using dry ice and isopropanol in 100 μL aliquots for lateruse.

To transfect, aliquots (40 μL) of thawed ARI814 cells were placed intoeach of three chilled microcentrifuge tubes. A peptide display librarybased on the undecamer carboxyl terminal peptide of Gαt (SEQ ID NO:15)was prepared according to Example 1. Two microliters of library plasmidwere added to the tubes and mixed. For the first round of “panning,” 200μl of the plasmid library was added. For subsequent rounds, three setsof transfections were performed (adherent plasmids from wells containingreceptor (+); adherent plasmids from wells containing no receptor (−);and the PRE sample which was not incubated). See below.

In each round of panning, less library was used (round 2:100 μl; round3:50 μl; round 4:10 μl). After the panning was completed, the DNA forthe LacI fusion protein was eluted. This DNA (50 μl) was used totransfect E. Coli cells by electroporation, using cold, sterile 0.1 cmelectrode gap cuvettes. The cuvettes were pulsed one time using a BioRadE. coli Pulsar set to 1.8 kV, 25 μF capacity, time constant 4-5mseconds, with the Pulser Controller unit at 200 mΩ. Immediately, 1 mLof SOC was added and the mixture transferred to a labeled 17×100 mmpolystyrene tube. The tube was shaken for one hour at 37° C. Aliquotswere taken from each set to plate 100 μL undiluted to 10⁻⁶ dilutionsamples on LB-Amp plates. Counts of the PRE plates indicated librarydiversity, while comparison of the (+) and (−) plates indicated whetherspecific clones were being enriched by the panning procedure.

The remaining ˜900 μL in the + receptor tube was added to a 1 L flaskcontaining 200 mL LB-AMP media, prewarmed to 37° C., and grown at 37°C., shaking until OD₆₀₀=0.5. The tube of cells then were placed in anice water bath for at least 10 minutes, and kept chilled at or below 4°C. during the subsequent washing steps. The cells were sedimented at5000×g for six minutes, resuspended in 100 mL WTEK buffer, sedimented at5000×g for six minutes, resuspended in 50 mL TEK buffer, resedimented at5000×g for six minutes and resuspended in 4 mL HEK buffer. The cellswere divided into the cryovials and stored at −70° C. One tube was usedfor the next round of panning and the other saved as a backup.

The panning process is illustrated in FIG. 1. For screening of thelibrary by “panning,” rhodopsin receptors prepared according to Example5 were immobilized directly on Immulon 4 (Dynatech) microtiter wells(0.1-1 μg of protein per well) in cold 35 mM HEPES, pH 7.5, containing0.1 mM EDTA, 50 mM KCl and 1 mM dithiothreitol (HEK/DTT). After shakingfor one hour at 4° C., unbound membrane fragments were washed away withHEK/DTT. The wells were blocked with 100 μl 2% BSA in HEKL (35 mM HEPES;0.1 mM EDTA; 50 mM KCl; 0.2 M α-lactose; pH 7.5, with 1 mM DTT). Forrounds 1 and 2, BSA was used for blocking; in later rounds 1% nonfat drymilk was used. For the first round of panning, about 24 wells of a96-well plate were used. In subsequent rounds, 8 wells with receptor and8 wells without receptor were prepared.

The Gt library was thawed (2 mL aliquot) and mixed with 6 mL lysisbuffer on ice. Lysis buffer contains 4.25 mL HE (25 mM HEPES: 0.1 mMEDTA; pH 7.5); 1 mL 50% glycerol; 750 μL 10 mg/mL protease-free BSA inHE; 10 μL 0.5 M DTT; and 6.25 μL 0.2 M PMSF. Freshly prepared lysozymesolution (150 μL 10 mg/mL lysozyme in cold HE) was added and the tubewas gently inverted several times and incubated on ice for no more thantwo minutes. The extent of lysis is evidenced by an increase inviscosity that can be observed by noting the slow migration of bubblesto the top of the tube after mixing. Lysis was terminated by mixing in 2mL 20% lactose and 250 μL 2M KCl. The tube was centrifuged immediatelyat 13,000×g for 15 minutes at 4° C. and the supernatant transferred to anew tube. A small aliquot of 0.1% (the PRE sample) was saved in aseparate, labeled tube. The blocked rhodopsin receptor-coated plate wasrinsed four times with HEKL/1% BSA and exposed to room light for lessthan five minutes on ice to activate the rhodopsin for light-activatedrhodopsin (Table IX), or left in the dark for dark-adapted (inactive)rhodopsin (Table X). Immediately thereafter, the crude bacterial lysatefrom the peptide library (200 μL) was added to each well and allowed toshake gently for one hour at 4° C. For round 2, this same procedure wasfollowed. In round 3, the amount of lysate used was reduced to 100 μL.In subsequent rounds, the lysate was diluted 1:10 in HEKL/BSA. In allrounds, 5-10 μL 200 μM native peptide was added to the wells to chaseoff peptides that were bound with lower affinity.

After incubation with the bacterial lysate, the wells were washed fourtimes into cold HEKL/1% BSA. Sonicated salmon sperm DNA (200 μL 0.1mg/mL in HEKL/1% BSA was added to each well and shaken gently for 30minutes at 4° C. The plates were washed four times with cold HEKL andtwice with cold HEK, then eluted by adding 50 μL/well 1 mM IPTG/0.2 MKCl in HE with vigorous shaking at room temperature for 30 minutes. Theeluants from each group of wells (+ or − receptor) were combined in oneor more microcentrifuge tubes as necessary. The volume of the PRE samplewhich had been saved previously was brought up to match the volume ofthe eluant samples and precipitated in parallel with them. Toprecipitate, 1/10 volume of 5M NaCl was mixed with each of the samples,then 1 μL 20 mg/mL glycogen was mixed with the samples. An equal volumeof RT isopropanol was then added and mixed thoroughly. The samples weresubjected to centrifugation at 13,000×g for 15 minutes and thesupernatant aspirated. The pellet was washed with 500 μL cold 80%ethanol and again subjected to centrifugation at 13,000×g for 10minutes. The pellets of plasmid DNA were resuspended in sterile,double-distilled water, 200 μL for the PRE sample and 4 μL for the + or− receptor samples and stored at −20° C.

Both light-activated rhodopsin and dark-adapted rhodopsin were used toscreen the library in this manner. See Tables IX and X, below. Six ofthe sequences obtained using light-activated rhodopsin were 100-1000times more potent than the native sequence at binding rhodopsin and arelisted in Table IX. When the Gαt library was used to pan light-activatedrhodopsin, residues L344, C347 and G348 were invariant. Also, in each ofthe highest affinity sequences, the basic residue at position 341 (R341)was changed to a neutral residue. When the Gαt library was used to pandark-adapted odopsin, the L344, C347 and G348 residues were no longervariant (L344 present in 62.5% of sequences, C347 present in 25% ofsequences, G348 present in 75% of sequences) and the residue at position341 was usually unchanged. Thus, the conformation of the receptor in itsinactive, dark-adapted state allows it to bind to a different set ofpeptide analogs than the light-activated receptor. In addition, itappears that in the light-activated receptor, it is the last seven aminoacids of the peptide which are most important (344-350) while the firstsix amino acids (340-345) are more important for dark-adapted rhodopsinbinding. TABLE IX Light-Activated Rhodopsin High Affinity Sequences.Clone No. SEQ ID NO: Sequence Library Sequence 124 IRENLKDCGLF 8 125LLENLRDCGMF 9 126 IQGVLKDCGLL 10 127 ICENLKECGLF 18 128 MLENLKDCGLF 23129 VLEDLKSCGLF 24 130 MLKNLKDCGMF 3 131 LLDNIKDCGLF 4 132 ILTKLTDCGLF 6133 LRESLKQCGLF 11 134 IHASLRDCGLF 13 135 IRGSLKDCGLF 14 136 IFLNLKDCGLF15/28 137 IRENLEDCGLF 16 138 IIDNLKDCGLF 17 139 MRESLKDCGLF 19 140IRETLKDCGLL 26 141 ILADVIDCGLF 27 142 MCESLKECGLF

TABLE X Dark-Adapted Rhodopsin High Affinity Sequences. Clone No. SEQ IDNO: Sequence Library Sequence 124 IRENLKDCGLF 2 143 IREKWKDLALF 3 144VRDNLKNCFLF 7 145 IGEQIEDCGPF 17 146 IRNNLKRYGMF 21 147 IRENLKDLGLV 26148 IRENFKYLGLW 33/37 149 SLEILKDWGLF 41 150 IRGTLKGWGLF

Example 8 Screens of PAR1 with a Gq Peptide Library

The methods of Example 7 were used to screen different sources of PAR1receptor using the Gq library. Purified PAR1, reconstituted in lipidvesicles (Example 6), membranes prepared from Sf9 insect cellsexpressing PAR1 (Example 3) and membranes prepared from mammalian cellsoverpressing PAR1 were used. The results of the screens are presented inTables XI, XII and XIII, respectively. The peptide used as thecompetitor for all three screens was LQLNLKEYNLV (SEQ ID NO:2). The4-residue linker sequences are random and are optionally present at theamino terminus of the binding peptide. These results show that theidentified high affinity peptides are similar for all three sources ofscreened PAR1. When a Gq-biased library is used to pan PAR1, thepositions that appear to be critical for receptor recognition, and thusare invariant, are N348, L349 and V350. TABLE XI Reconstituted PurifiedRecombinant PAR1 Receptor; Screening Results. SEQ ID Clone Linker SEQ IDNO: LQLNLKEYNLV NO:2 R2-16 *SWV 151 LQFNLNDCNLV 102 R2-17 FVNC 152LQRNKKQYNLG 160 R2-18 EVRR 153 MKLKLKEDNLV 103 R2-20 *RVQ 154HQLDLLEYNLG 104 R2-21 RLTR 155 LQLRYKCYNLV 161 R3-37 SR*K 156LQQSLIEYNLL 111 R3-38 MTHS 157 VHVKLKEYNLV 162 R3-44 SGPQ 158LQLNVKEYNLV 163 R3-46 ML*N 159 LRIYLKGYNLV 164

TABLE XII PAR1 Receptor Sf9 Insect Cell Membranes; Screening Results.SEQ ID Clone Linker SEQ ID NO: LQLNLKEYNLV NO:2 S1-13 S*IR 165MKLNVSESNLV 94 S1-18 RWIV 166 LQLNLKVYNLV 175 S1-23 G*GH 167 LELNLKVYNLF176 S2-26 RSEV 168 LQLKHKENNLM 100 S2-30 CEPG 169 LHLNMAEVSLV 177 S2-36HQMA 170 LQVNLEEYHLV 101 S3-6  VPSP 171 LQKNLKEYNMV 106 S3-8  QMPN 172LQMYLRGYNLV 108 S3-10 MWPS 173 LKRYLKESNLV 178 S3-15 C*VE 174MNLTLKECNLV 110

TABLE XIII Mammalian (CHO) Cells Overexpressing PAR1; Screening Results.SEQ ID Clone Linker SEQ ID NO: LQLNLKEYNLV NO:2 C4-5  PRQL 179LQLKRGEYILV 183 C4-19 VRPS 3 LQLNRNEYYLV 4 C5-10 SRHT 11 LRLNGKELNLV 12C5-12 FFWV 180 CSLKLKAYNLV 184 C4-16 QRDT 181 LQMNHNEYNLV 185 C7-3  NERN182 PQLNLNAYNLV 186 C7-10 LPQM 9 QRLNVGEYNLV 10 C7-13 LSTN 7 LHLNLKEYNLV8 C7-14 LSRS 5 LQQKLKEYSLV 6

Example 9 Identification of GPCR-Binding High Affinity Peptide Analogs(Panning)

The methods of Example 7 were repeated using recombinant reconstitutedβ₂ adrenergic receptor panned with the Gs Library. Results of thepanning screens and ELISA binding affinity of the selected peptides areshown in Table XIV, below. TABLE XIV β2-Adrenergic Receptor screenedwith Gs library. SEQ ID NO Competitor QRMHLRQYELL 13 ELISA AG1QGMQLRRFKLR 187 .435 AG20 RWLHWQYRGRG 188 .431 AG19 PRPRLLRFKIP 189 .361AG2 QGEHLRQLQLQ 190 .330 AG4 QRLRLGPDELF 191 .291 BAR1 QRIHRRPFKFF 192.218 AG3 QRMPLRLFEFL 193 .217 BAR2 QRVHLRQDELL 194 .197 AG11 DRMHLWRFGLL195 .192 AG9 QRMPLRQYELL 196 .190 BAR3 QWMDLRQHELL 197 .185 AG18QRMNLGPCGLL 198 .155 BAR20 NCMKFRSCGLF 199 .079 AG13 QRLHLRGYEFL 200.054 BAR11 HRRHIGPFALL 201 .048 BAR8 ERLHRRLFQLH 202 .047 BAR40PCIQLGQYESF 203 .028 BAR31 QRLRLRKYRLF 204 .026

Example 10 Identification of GPCR-Binding High Affinity Peptide Analogs(Panning)

The methods of Example 7 repeated using rhodopsin screening with a Gtlibrary. Results of the panning screens and ELISA binding affinity ofthe selected peptides are shown in Table XV, below. To identify the rankorder of binding, the lysates were analyzed using ELISA methods in whichthe secondary antibody was conjugated to HRP. Following addition of thesubstrate, the microplate was read using a spectrophotometer. Thebinding is the OD₄₅₀ for wells with receptor—OD₄₅₀ for wells in which noreceptor (control wells with empty lipid vesicles). TABLE XV Rhodopsinscreened with Gt library. SEQ ID NO: Competitor IRENLKDCGLF 124 ELISAL33 IVEILEDCGLF 205 1.007 L4 MLDNLKACGLF 206 .908 L3 ILENLKDCGLF 207.839 L14 LRENLKDCGLL 208 .833 L38 LLDILKDCGLF 209 .823 L15 VRDILKDCGLF210 .621 L34 ILESLNECGLF 211 .603 L17 ILQNLKDCGLE 212 .600 L7MLDNLKDCGLF 213 .525 L10 IHDRLKDCGLF 214 .506 L20 IRGSLKDCGLF 135 .423L6 ICENLKDCGLF 215 .342 L8 IVKNLEDCGLF 216 .257 L13 ISKNLRDCGLL 217 .187L10 IRDNLKDCGLF 218 .162

Example 11 Additional Peptide Analogs

Chinese hamster ovary-expressed PAR1 was screened against the Gt, G12and G13 libraries, using the competitor peptide indicated in Table XVIbelow. Additional peptide analogs were identified using the Gt, G12 orG13 library as indicated and IRENLKDCGLF (SEQ ID NO:124), LQENLKDIMLQ(SEQ ID NO:38) or LQDNLKQLMLQ (SEQ ID NO:233), respectively ascompetitor with screening for high affinity binding to PAR1 receptorobtained from Chinese hamster ovary cells as described in Example 1,indicated in Table XVII, below. TABLE XVI Peptides Identified with CHOEXPRESSED PAR1. Gt library G12 library G13 library (IRENLKDCGLF;(LQENLKDIMLQ; (LQDNLKQLMLQ; SEQ ID NO:124) SEQ ID NO:38) SEQ ID NO:233)IREFLTDCGLF 219 LQENLKEMMLQ 225 LQDNLRHLMLQ 234 IRLDLKDVSLF 220LEENLKYRMLD 226 LQDKINHLMLQ 235 ICERLNDCGLC 221 LQEDLKGMTLQ 227LQANRKLGMLQ 236 PRDNTKVRGLF 222 LQETMKDQSLQ 228 LIVKVKQLIWQ 237FWGNLQDSGLF 223 PQVNLKSIMRQ 229 MRAKLNNLMLE 238 RRGNGKDCRHF 224WQHKLSEVMLQ 230 LQDNLRHLIQ 239 LKEHLMERMLQ 231 LQDNRNQLLF 240LLGMLEPLMEQ 232

TABLE XVII PAR1 Binding Peptides Screened using a G11 Library(LQLNLKEYNLV; SEQ ID NO: 2) SF9 CHO EXPRESSED SEQ ID NO: Recomb/ReconstSEQ ID NO: EXPRESSED SEQ ID NO: LQLNVKEYNLV 163 LQLNVKEYNLV 163LQLNLKVYNLV 175 LQLNRKNYNLV 241 LQLRVKEYKRG 244 LQLKHKENNLM 100LQLRYKCYNLV 161 LQLRYKCYNLV 161 LQKNLKEYNMV 106 LQLDLKESNMV 242LQIYLKGYNLV 245 LQVNLEEYHLV 101 LQLNLKKYNRV 243 LQFNLNDCNLV 102LFLNLKEYSLV 257 LQLRVKEYKRG 244 LQRNKKQYNLG 160 LELNLKVYNLV 258LQRNKKQYNLG 160 LQRNKNQYNLG 254 LPLNPKEYSLV 109 LQIYLKGYNLV 245LQQSLIEYNLL 111 LPLNLIDFSLM 259 LQFNLNDCNLV 102 LRLDFSEKQLV 105LPRNLKEYDLG 260 LQYNLKESFVV 246 LYLDLKEYCLF 255 LRLNDIEALLV 261LQQSLIEYNLL 111 HQLDLLEYNLG 104 LVLNRIEYNLL 262 LQRDHVEYKLF 247VQVKLKEYNLV 251 LHLNMAEVSLV 177 LVIKPKEFNLV 248 MKLKLKEDNLV 103MNLTLKECNLV 110 IQLNLKNYNIV 249 SAKELDQYNLG 256 MKLNVSESNLV 94HQLDLLEYNLG 104 VHVKLKEYNLV 162 LKRYLKESNLV 178 MQLNLKEYNLV 250LKRKLKESNMG 263 VQVKLKEYNLV 251 LKRKVKEYNLG 264 QLLNQYVYNLV 252LELNLKVYNLF 176 MKLKLKEDNLV 103 LQMYLRGYNLV 108 WRLSLKVYNLV 253LQLKRGEYILV 183 LQLNRNEYYLV 4 LRLNGKELNLV 12 CSLKLKAYNLV 184 LQMNHNEYNLV185 PQLNLNAYNLV 186 QRLNVGEYNLV 10 LHLNLKEYNLV 8 LQQKLKEYSLV 6

Example 12 Preparation of LacI Lysates

In the last round of panning, several clones were selected from the (+)receptor plates and grown up overnight in LB-Amp media. Three hundredmicroliters of the overnight culture was diluted in 3 mL LB-Amp mediafor “ELISA lysate culture.” Another 30 μL was added to an equal volumeof 50% glycerol was stored in labeled microcentrifuge tubes at −70° C.The remaining 4.5 mL was used to make DNA using a standard miniprepprotocol (Qiagen Spinprep™ kits) and sequenced using a 19 base pairreverse primer which is homologous to the vector at a site 56 basepairsdownstream from the TAA stop codon that terminates the random region ofthe library (GAAAATCTTCTCTCATCCG; SEQ ID NO:265). The DNA was stored at−20° C. The ELISA lysate culture was allowed to shake for one hour at37° C. Expression was induced by adding 33 μL 20% arabinose (0.2% finalconcentration) with shaking at 37° C. for 2-3 hours. The culture thenwas subjected to sedimentation at 4000×g for five minutes, the pelletresuspended in 3 mL cold WTEK buffer, resedimented at 4000×g for fiveminutes and the pellet resuspended in 1 mL cold TEK buffer. Aftertransfer to 1.5 mL microcentrifuge tubes, the pellet was sedimented at13,000×g for two minutes and the supernatant aspirated. The cell pelletwas resuspended in 1 mL lysis buffer (42 mL HE, 5 mL 50% glycerol, 3 mL10 mg/mL BSA in HE, 750 μL 10 mg/mL lysozyme in HE and 62.5 μL 0.2 MPMSF) and incubated on ice for one hour. One hundred ten microliters 2MKCl was added to the lysis mixture and inverted to mix, then sedimentedat 13,000×g for 15 minutes at 4° C. The clear crude lysate (about 0.9 mLsupernatant) was transferred to a new tube and stored at −70° C.

Example 13 PAR1 Receptor-Specific Binding of LacI-Peptide FusionProteins

The binding properties of the peptide encoded by individual clones wereassayed as follows. Purified PAR1 receptor prepared from Sf9 insectcells (1-10,000 pg/mL in 50 mM Tris-HCl, pH 7.4, 10% glycerol) wasreconstituted in lipid vesicles according to Example 6. A serialdilution of the membranes containing receptor ranging from 0.2 to 20,000μg/mL (+/− receptor) was added to wells on a microtiter plate and shakengently for one hour at 4° C. After washing, a 1:1 to 1:10,000 serialdilution of a LacI-Gq lysate prepared from the LacI-Gq clone accordingto the methods described in Example 12 was added to the wells, the platewas shaken gently for one hour at 4° C., and washed. Anti-LacIantibodies (Stratagene) were added (1:1000) and the plate shaken gentlyfor one hour at 4° C. After washing, HRP-conjugated goat anti-rabbitantibodies (Kierkegaard and Perry Laboratories) were added (1:2500) andthe plate shaken gently for one hour at 4° C. The plate was washed,color was developed using horseradish peroxidase, and then read in anELISA reader at OD₄₅₀. The general methodology for the ELISA isillustrated in FIG. 4. The results, see FIG. 5, show that the LacI-Gqfusion protein binds thrombin receptor in a concentration-dependentmanner. The ability of the LacI-Gq fusion protein to bind the emptyvesicles was significantly less than vesicles reconstituted withthrombin receptor.

Example 14 Screening in the Presence of a High Affinity Peptide

To identify peptides having even higher affinity to light-activatedrhodopsin than those identified by the panning procedure described inExample 7, a high affinity peptide was included in the libraryincubations in rounds three and four. Peptide 8 (LLENLRDCGMF; SEQ IDNO:125) had been identified in the first screening as a peptideexhibiting binding to light-activated rhodopsin 1000-fold higher thanthe native sequence. Screening of the Gαt library was performed as inExample 7, except that 10 μL 100 μM (100 nM final concentration) peptide8 was included in the wells in rounds three and four. This screenrevealed several clones that both bind rhodopsin with very high affinityand stabilize it in its active form, metarhodopsin II. See Table XVIII,below. Comparing Tables IX and XVIII, it is clear using peptide 8 in thescreen resulted in a change at position 341 to a neutral residue.Residues L344, C347 and G348 remained stable whether peptide 8 wasincluded in the screen or not. Use of peptide 8 resulted in a higherincidence of isoleucine at position 340 (17% with native peptide versus71% with peptide 8) and a lower incidence of glutamine at position 342(67% with native peptide versus 29% with peptide 8) This type ofinformation not only contributes to the discovery of highly potentanalog peptides for use as drugs or drug screening compounds, but alsofurthers the understanding of the structural framework which underliesthe sites of contact between Gα and receptor.

Binding assays performed on some of the clones identified in this wayare shown in FIG. 6. All peptides identified using peptide 8 in thescreening process bound with equal or greater affinity tolight-activated rhodopsin as did peptide 8. Compare the first bar(HAP=peptide 8) with the remaining bars. TABLE XVIII ExemplaryLight-Activated Rhodopsin High Affinity Sequences Identified in Screenswith Addition of Peptide 8. Clone No. SEQ ID NO: Sequence LibrarySequence 124 IRENLKDCGLF Peptide 8 125 LLENLRDCGMF 3 266 ILENLKDCGLL 7213 MLDNLKDCGLF 8 216 IVKNLEDCGLF 10 218 IRDNLKDCGLF 13 217 ISKNLRDCGLL17 212 ILQNLKDCGLF 19 206 MLDNLKACGLF

Example 15 Subcloning into MBP Vectors and Preparation of MBP CrudeLysates

pELM3 was digested at room temperature with AgeI (New England Biolabs)and the cut vector was separated from uncut vector on a 0.7% agarosegel. DNA was purified (Qiagen Extract-a-gel kit) and digested with ScaI(New England Biolabs). The 5.6 kb MBP vector fragment was separated on a1% agarose gel and purified as above. During the final affinitypurification round of the peptide library, a 20 mL portion of the 200 mLamplification culture was set aside before harvesting the cells. This 20mL portion was allowed to grow to saturation, usually overnight, and DNAwas prepared from the cells (Qiagen midi-prep kit). The pJS142 plasmidDNA was digested with BspEI and ScaI. The 0.9 kb peptide-encodingfragment was separated from the 3.1 and 1.7 kb vector fragments on a 1%agarose gel and purified.

Different ratios of the 5.6 kb MBP vector fragment and thepeptide-encoding 0.9 kb fragment (1:2, 1:1, 2.5:1, 5:1, 10:1) wereligated in ligase buffer containing 0.4 mM ATP at 14° C. overnight withT4 DNA ligase. The ligation was terminated by increasing the temperatureto 65° C. for ten minutes. To lower the background, the ligation mixturewas digested with XbaI before isopropanol precipitation using 1 μLglycogen as a carrier. After one wash with 80% ethanol, the pellet wasresuspended in 20 μL double-distilled water. ARI814 cells weretransformed as described in Example 7 using 1 μL of the precipitatedXbaI digested ligation mix. After allowing the cells to shake for onehour at 37° C. in 1 mL SOC, 100 μL of the suspension was spread onLB-Amp Plates. Crude lysates were prepared as described for LacI lysatesin Example 12.

Example 16 MBP-Peptide Fusion Protein Purification

An overnight culture (1 mL) of a single MBP-peptide fusion protein clonewas inoculated into 200 mL LB-AMP media. The culture was shaken at 37°C. until OD₆₀₀=0.5. Protein expression was induced by addition of 150 μL1 M IPTG (final concentration 0.3 mM), with continued shaking at 37° C.for two hours. The culture then was sedimented at 5000×g for 20 minutesand resuspended in 5 mM column buffer (10 mM Tris, pH 7.4; 200 mM NaCl;1 mM EDTA; 1 mM DTT) and 16.25 μL 0.2 M PMSF was added. The resuspendedcell pellet was then stored at −70° C. The stored pellet was thawed incold water and placed in an ice bath. The pellet was sonicated in shortpulses of less than 15 seconds with a Fisher Scientific 55 SonicDismembrator (40% constant time, output 5, repeating five times with atotal one minute duration). The sonicated pellet was subjected tocentrifugation at 9000×g for 30 minutes, after which the supernatant wassaved and diluted to 100 mL using column buffer. Usually, the proteinconcentration was approximately 2.5 mg/mL. A column was prepared bypouring 7.5 ml amylose resin in a BioRad disposable column and washingwith eight volumes of column buffer. The diluted crude extract wasloaded by gravity flow at about 1 mL/min and the column was washed againwith eight volumes of column buffer. The fusion protein was eluted with10 mL 10 mM maltose in column buffer and concentrated using Amiconcentriplus 30™ columns, then aliquoted and stored at −70° C.

Example 17 Method for Screening Library Crude Lysates by ELISA

Microtiter wells were coated with 0.1-1.0 μg/well rhodopsin receptor ina final volume of 100 μL HEK containing 1 mM DTT with shaking at 4° C.for one hour. The wells then were blocked with bovine serum albumin(BSA) by adding 100 μL 2% BSA in HEK with 1 mM DTT to the wells andcontinuing shaking at 4° C. for at least 30 minutes, then washed fourtimes with HEK containing 1 mM DTT. Crude lysates were diluted 1:50 inHEK containing 1 mM DTT and added to the coated wells (100 μL per well).The plates were shaken at 4° C. for one hour, washed four times withPBS/0.05% Tween™20 1 mM maltose and then probed with 100 μL 1:1000rabbit anti-MBP antibodies (New England BioLabs) in PBS containing 0.05%Tween™ 20 and 1 mM maltose, with shaking for 30 minutes at 4° C. Afteranother wash, the wells were probed with 100 μL 1:7500 goat anti-rabbitsecondary antibodies conjugated to horseradish peroxidase in PBScontaining 1% BSA and 1 mM maltose with shaking for 30 minutes at 4° C.The plate was washed four times with PBS containing 0.05% Tween™ 20 and1 mM maltose. Horseradish peroxidase substrate (Bio-Fx; 100 μL) wasadded and the color developed for 20-30 minutes. The reaction wasstopped by addition of 100 μL 2N sulfuric acid and the plate read atOD₄₅₀. If the color reaction occurred too quickly (less than 10 minutes)or if the background in negative control wells was too high (greaterthan 0.2) the assay was repeated using 1:100 or 1:200 dilutions of thecrude lysates.

Example 18 Binding Assay of High Affinity Rhodopsin Binding PeptideFusion Proteins

The entire population of peptide-coding sequences identified in round 4of panning (see Example 7) was transferred from pJS142 to pELM3 (NewEngland Biolabs). This plasmid is a pMal-c2 derivative with a modifiedpolylinker, inducible by isopropyl β-thiogalacto-pyranoside andcontaining the E. coli malE gene with a deleted leader sequence andleads to cytoplasmic expression of MBP fusion proteins. The MBP-carboxylterminal peptide analog fusion proteins were expressed in E. coli.

For the assay, in the dark, 1 μg/well of ROS membranes (rhodopsin) asdescribed in Example 5 was directly immobilized on microtiter wells incold HEK/DTT for one hour at 4° C. The wells were rinsed, blocked with1% BSA in HEK/DTT for one hour at 4° C. and rinsed again. Boundrhodopsin was activated by exposure to light for 5 minutes on ice beforeaddition of the MBP fusion proteins (crude bacterial lysates werediluted 1:50 in HEK with 1 μM dithiothreitol; purified proteins wereused at 0.2-120 nM). The MBP-Gαt340-350K341R (pELM17) fusion protein andMBP with linker sequence only (pELM6) were present in control wells at50 nM final concentration. After 30 minutes, wells were washed andrabbit anti-MBP antibody (New England Biolabs) was added. The anti-MBPantibody was used at a 1:1000 dilution for crude lysates and a 1:3000dilution for purified proteins. After 30 minutes, wells were rewashedand goat anti-rabbit antibody conjugated to horseradish peroxidase(1:7500 dilution for crude lysates; 1:10,000 dilution for purifiedproteins; Kierkagaard & Perry Laboratories) was added. After 30 minutes,the plate was washed four times with PBS containing 0.05% Tween™20.Horseradish peroxidase substrate (100 μl) was added and color wasallowed to develop for about 20 minutes. The reaction was stopped byaddition of 100 μl 2N sulfuric acid. The results are presented in FIG.7. Values indicate absorbance at OD₄₅₀. The positive control for theassay was pELM 17, which encodes the MBP fusion proteinGα_(t)340-350K341R. pELM6, which expresses MBP protein fused to a linkersequence only, served as the negative control. “No lysate” control wellswere included to reflect any intrinsic, non-specific binding within theassay. See FIG. 7.

The IC₅₀ values of the high affinity MBP fusion proteins ranged from 3.8to 42 nM, up to 3 orders of magnitude more potent than the 6 μM IC₅₀ ofMBP-Gαt340-350K341R. In all the highest affinity sequences, position341, which is a positively charged residue in the native sequence, waschanged to a neutral residue. Leu344, Cys347, and Gly348 were found tobe invariant and hydrophobic residues were always located at positions340, 349, and 350, indicating the critical nature of these residues.

Example 19 Binding of High Affinity Peptide Fusion Proteins to RhodopsinCan Be Competitively Inhibited by Heterotrimeric Gt

When light-activated rhodopsin was screened for peptides based on theC-terminus of Gt, a large number of high-affinity sequences wereobtained. Binding of MBP fusion proteins containing the high affinitypeptide from the library (sequences from clones 8, 9, 10, 18, 23, 24, aswell as pELM17 which encodes the wild-type peptide sequence, and pELM6which contains no peptide; MBP-8, MBP-9, MBP-10, MBP-18, MBP-23, MBP-24,MBP-pELM17) were assessed for their ability to bind rhodopsin (0.5 μgrhodopsin/well) in the presence or absence of heterotrimeric Gt.

Lysate (50 μl) from each clone was added and incubated in the light.After 45 minutes, 1 μM heterotrimeric Gt was added and the solutionincubated for 30 minutes. Anti-MBP antibody was added, followed by goatanti-rabbit alkaline phosphatase conjugated antibody and substrate. Thecolor was allowed to develop. Absorbance data are presented in FIG. 8.

Most peptide sequences obtained were highly homologous to the native GαtC-terminal sequence. Several of these sequences are of very highaffinity (>1000-fold higher than the parent peptide) and are potent andspecific antagonists of receptor-mediated G protein activation. Thehigh-affinity peptide fusion proteins were tested for binding tolight-activated rhodopsin and for their ability to stabilize the MIIconformation (Table XXI).

The screen used MBP-8 because this peptide bound to rhodopsin with highaffinity and stabilized MII. MBP-18 and MBP-24, which both showed evenhigher binding affinities than did MBP-8 to rhodopsin, were not usedbecause the affinity was so high that the small molecules might not havebeen able to competitively inhibit their binding. Of course, the screenmay be repeated using another peptide as is convenient, for examplepeptides that are of higher affinity to find even more potent smallmolecules. TABLE XIX Absorbance at OD₄₅₀ in a Panning ELISA and EC50values for MII binding and MII Stabilization for Selected MBP-HighAffinity Peptide Fusion Proteins. SEQ MII MII ID ELISA bindingstabilization NO: OD₄₅₀ EC₅₀ EC₅₀ Gt IKENLKDCGLF 15 .01 6000 nM >100 μM9 LQQVLKDCGLL 267 .35 10 nM 1.05 μM 10 ICENLKDCGLF 215 .36 42 nM 5.40 μM8 LLENLRDCGMI 268 .54 7.8 nM 0.94 μM 18 MLENLKDCGLF 128 .58 3.8 nM 1.24μM 24 MLKNLKDCGMF 130 .61 6.6 nM 0.49 μM 23 VLEDLKSCGLF 129 .66 20 nM3.50 μM

Heterotrimeric Gt competitively inhibited high affinity peptide fusionprotein binding to light-activated rhodosin. See FIG. 8. Theheterotrimeric Gt contains multiple determinants of rhodopsin bindingand is membrane bound via myristoylation of the α subunit andfarnesylation of the γ subunit carboxyl terminus. Thus, the selectedpeptide sequences from the combinatorial library bind to the receptorwith very high affinity.

Example 20 Binding of MBP Clones to PAR1

To identify high affinity peptides that bind PAR1, membranes preparedfrom mammalian cells (Chinese hamster ovary) overexpressing PAR1 werepanned with the G11 peptide library. ELISA binding affinity results ofselected clones are shown in FIG. 9 for their binding to membranesprepared from SF9 cells expressing either PAR1 or the Gq-coupledmuscarinic M1 receptor. To quantitate the binding, purified MBP cloneswere analyzed using ELISA methods in which the secondary antibody wasconjugated to HRP. The binding for the control MBP-Gq fusion protein isshown. See FIG. 9. The data are the average of two separate experimentsdone in duplicate. MBP clones PAR-13 and PAR-34 both show both highaffinity binding for PAR1 as well as specificity. MBP clones PAR-23 andPAR-33 appear to be both of low affinity and low specificity. See TableXIII for the sequences.

Example 21 Binding Specificity of LacI-Peptide Fusion Proteins

PAR1 binding clones of LacI-peptide fusion protein selected from the G11Library were diluted 1:100 in HEK/DTT and tested for dose-responsivebinding to Sf9 insect cell membranes from cells expressing no receptor,the M1 receptor (which couples to Gi) or PAR1 receptor, preparedaccording to Example 3. Increasing amounts of membrane as indicated inFIG. 10 were coated in microtiter wells, incubated and rinsed.LacI-peptide fusion protein lysates were added, incubated and rinsed,and the receptor-bound LacI-peptide fusion protein was measured asdescribed above using a LacI antibody. Results for a single,representative clone are presented in FIG. 10, and demonstrate thespecificity of the selected peptides for PAR1.

Example 22 Binding of Native Gαq-Maltose Binding Protein-Peptide FusionProtein to PAR1

Microtiter wells were coated with purified, reconstituted PAR1 in thepresence of 100 nmoles thrombin receptor activating peptide, asdescribed above in Example 6. Purified maltose binding protein-Gαq(MBP-Gαq) was added at the concentrations indicated in FIG. 11 andincubated one hour on a shaker at 4° C. The wells were rinsed and thenprobed with a rabbit anti-maltose binding protein antibody, followed byalkaline phosphatase conjugated secondary antibodies, as describedabove. Substrate was added and the color was allowed to develop about 20minutes. Absorbance at 405 nm was measured and dose-response curves werecalculated using GraphPad Prism (version 2.0). See results in FIG. 11.The calculated IC₅₀ of Gαq binding to activated PAR1 was 214 nM.

Example 23 Design of Oligonucleotides for Gα Peptide Minigene Constructs

cDNA encoding the last 11 amino acids of Gα subunits was synthesized(Great American Gene Company) with newly engineered 5′- and 3′-ends. The5′-end contained a BamHI restriction enzyme site followed by the humanribosome-binding consensus sequence (5′-GCCGCCACC-3′; SEQ ID NO:269), amethionine codon (ATG) for translation initiation, and a glycine codon(GGA) to protect the ribosome binding site during translation and thenascent peptide against proteolytic degradation. A HindIII restrictionenzyme site was synthesized at the 3′ end immediately following thetranslational stop codon (TGA). Thus, the full-length 56 bpoligonucleotide for the Giα_(1/2) carboxyl terminal sequence was5′-gatccgccgccaccatgggaatcaagaacaacctgaaggactgcggcctcttctgaa-3′ (SEQ IDNO:270) and the complimentary strand was5′-agctttcagaagaggccgcagtccttcaggttgttcttgattcccatggtggcggcg-3′ (SEQ IDNO:271). See FIG. 12. As a control, oligonucleotides encoding theGαi_(1/2) carboxyl terminus in random order (GαiR) with newly engineered5′- and 3′-ends also were synthesized.

The DNA was diluted in sterile ddH₂O to form a stock concentration at100 μM. Complimentary DNA was annealed in 1× NEBuffer 3 (50 mM Tris-HCl,10 mM MgCl₂, 100 mM NaCl, 1 mM DTT; New England Biolabs) at 85° C. for10 minutes then allowed to cool slowly to room temperature. The DNA thenwas subjected to 4% agarose gel electrophoresis and the annealed bandwas excised. DNA was purified from the band using a kit, according tothe manufacture's protocol (GeneClean II Kit, Bio101). After digestionwith each restriction enzyme, the pcDNA 3.1(−) plasmid vector wassubjected to 0.8% agarose gel electrophoresis, the appropriate band cutout, and the DNA purified as above (GeneClean II Kit, Bio101). Theannealed/cleaned cDNA was ligated for 1 hour at room temperature intothe cut/cleaned pcDNA 3.1 μlasmid vector (Invitrogen) previously cutwith BamHI and HindIII.

For the ligation reaction, several different ratios of insert to vectorcDNA (ranging from 25 μM:25 pM to 250 pM:25 pM annealed cDNA) wereplated. Following the ligation reaction, the samples were heated to 65°C. for 5 min to deactivate the T4 DNA ligase. The ligation mixture (1μl) was electroporated into 50 μl competent cells as described inExample 7 and the cells immediately placed into 1 ml of SOC (Gibco).After 1 hour shaking at 37° C., 100 μl of the electroporated cellscontaining the minigene plasmid DNA was spread on LB/Amp plates andincubated at 37° C. for 12-16 hours. To verify that insert was present,colonies were grown overnight in LB/Amp and their plasmid DNA purified(Qiagen SpinKit). The plasmid DNA was digested with Ncol (New EnglandBiolabs, Inc.) for 1 hour at 37° C. and subjected to 1.5% (3:1) agarosegel electrophoresis. Vector alone produced 3 bands. When the 56 bpannealed oligonucleotide insert is present, there is a new NcoI siteresulting in a shift in the band pattern such that the digest patterngoes from three bands (3345 bp, 1352 bp, 735 bp) to four bands (3345 bp,1011 bp, 735 bp, 380 bp). See FIG. 13. DNA with the correctelectrophoresis pattern was sequenced to confirm the appropriatesequence. This method may be used to insert any high affinity peptide tocreate a minigene constant.

Example 24 Expression of Peptides from Minigene Constructs

Expression of the GPCR binding peptides was achieved using constructswhich included minigene inserts corresponding to the carboxyl terminalsequences of various G protein α subunits (Gαi, Gαo, Gαs, Gαq, Gα11,Gα12, Gα13, Gα14), as well as a control minigene containing the Gαisequence in random order (GαiR). The minigene insert DNAs were made bysynthesizing short complimentary oligonucleotides corresponding to thepeptide sequences from the carboxyl terminus of each Gα with BamHI andHindIII restriction sites at the 5′ and 3′ ends, respectively.Complementary oligonucleotides were annealed and ligated into themammalian expression vector pcDNBA3.1 according to the methods ofGilchrist et al., J. Biol. Chem. 274:6610-6616, 1999, the disclosures ofwhich are hereby incorporated by reference.

Human embryonic kidney (HEK) 293 cells were transfected using a standardcalcium phosphate procedure according to the methods of Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York, vol. 1-3 (1989), the disclosures of which are herebyincorporated by reference. To confirm the transcription of minigeneconstructs in transfected cells, total RNA was isolated from the cells48 hours post transfection with pcDNA-Gαi or pcDNA-GαiR using methodsknown in the art. Reverse transcriptase PCR was used to make cDNA andPCR analysis was performed using the cDNA as template with primersspecific for the relevant Gα carboxyl terminal peptide insert (forward:5′-ATCCGCCGCCACCATGGGA (SEQ ID NO:272); reverse: 5′-GCGAAAGGAGCGGGGCGCTA(SEQ ID NO:273)). These primers for the Gα minigenes amplify a 434 bpfragment only if the inserted peptide-encoding oligonucleotides arepresent; no band is observed in cells transfected with the emptypcDNA3.1 vector. The PCR products were separated on 1.5% agarose gels.The presence of a single 434 bp band indicated that Gα carboxyl terminuspeptide minigene RNA had been transcribed. See FIG. 14. Controlexperiments were done using a T7 forward primer with the vector reverseprimer to verify the presence of the pcDNA3.1 vector, and G3DPH primers(Clonetech) to approximate the amount of total RNA.

To verify that the peptide was being produced in the transfected cells,the cells were lysed and homogenized 48 hours post transfectionaccording to known methods. Cytosolic extracts were analyzed by gradientreversed phase HPLC as follows: 100 μL of cytosolic fraction extract wasloaded onto a C4 column (Vydac) equilibrated with 0.1% TFA in ddH₂O. Thepeptide was eluted using 0.1% TFA in an acetonitrile gradient whichincreased from 0-60% over 45 minutes. Peaks were collected, lyophilized,and analyzed using ion mass spray analysis (University ofIllinois-Urbana Champagne). Mass spectrometry analysis for peak 1 fromGα_(1/2) peptide vector (pcDNA-Gαi) transfected cells, and from cellstransfected with pcDNA-GαiR indicated that a 1450 Dalton peptide (theexpected molecular weight for both 13 amino acid peptide sequences) waspresent in each cytosolic extract. The minigene-encoded peptides werethe major peptides found in the cytosol, strongly indicating that thevectors produced the appropriate peptide sequences in large amounts.

Example 25 Interfacial G Protein Peptide Inhibition of Thrombin-MediatedInositol Phosphate Accumulation

HMEC were seeded onto 6-well plates 24 hours before transfection at1×10⁵ well. Cells were transiently transfected with pcDNA3.1, pcDNA-Gαi,pcDNA-GαiR, or pcDNA-Gq as described in Example 24. After 24 hours,cells were incubated in 2 mL culture medium containing 4 μCi/mL[³H]-myoinositol to obtain steady-state labeling of cellular inositollipids. Transiently transfected cells were assayed for inositolphosphate (IP) accumulation 48 hours after transfection. Two hours priorto stimulation with α-thrombin, cells were washed, and medium replacedwith medium containing 5 mM LiCl. Cells were stimulated with 10 nMα-thrombin for 10 minutes. Inositol phosphate (IP) formation was stoppedby aspiration of the medium and addition of ice-cold methanol (finalconcentration 5%).

Perchloric acid-lysed cells were centrifuged at 2500 rpm, 4° C. for 5min. The supernatant containing IP was eluted through a Poly-Prepchromatography column (Bio-Rad) containing 1.6 ml anion exchange resin(DOWEX AG1-X8, formate form, 200-400 mesh). The perchloricacid-precipitated pellets (containing phosphatidylinositols and lipids)were resuspended in 1 ml chloroform-methanol-10 M HCl (200:100:1,v/v/v). These suspensions were mixed with 350 μL HCl and 350 μLchloroform and sedimented for 5 min at 2500 rpm to separate the phases.The lower, hydrophobic phase was recovered and dried in counting vialsto determine the amount of radioactivity in total phosphatidylinositols.The relative amount of [³H]-IP generated was calculated as follows:([³H]-IP (cpm)/[³H]-IP (cpm)+[³H]-inositol (cpm)). Each value wasnormalized using the basal value (no thrombin stimulation) obtained inpcDNA transfected cells. See FIG. 15. The results presented are thenormalized mean±SEM of at least 3 independent experiments performed intriplicate. The ** symbol indicates p<0.005. Results indicate thataddition of thrombin increased IP production in control cells (pcDNA,pcDNA-GiR). Cells transfected with PcDNA-Gq had no thrombin-mediated IPproduction increase, while cells transfected with pcDNA-Gi had a normalresponse. This indicates that transfection of the Gq C-terminal minigenevector into HMEC with subsequent expression of the Gq C-terminal peptidecan inhibit thrombin-mediated increases in IP.

Example 26 Interfacial G Protein Peptide Inhibition of Thrombin-InducedP1 Hydrolysis and Intracellular Ca⁺⁺ Rise

To determine whether expression of the Gαq C-terminal minigene vectorcould affect intracellular [Ca⁺⁺]_(i) levels, HMEC were transfected withempty vector (pcDNA) or with pcDNA-Gαi, pcDNA-Gαq, or pcDNA-GαiRminigene DNA (1 μg), which encode high affinity peptides identified fortheir ability to bind the receptors. Transfected cells were seeded at alow confluency on coverslips in a 24-well plate 48 hours posttransfection. The cells were allowed to adhere for two hours. The mediumwas aspirated and each coverslip was incubated with 10 μM Oregon Green488 BAPTA-1 acetoxymethyl ester (a calcium-sensitive dye) and 0.1% (v/v)Pluronic F-127 and allowed to incubate for 20-30 minutes at 37° C., thenrinsed twice with wash buffer. Basal conditions were established beforeaddition of thrombin (˜70 nM) in Ca⁺⁺ buffer. Recordings were made every10 seconds and continued for 170 seconds after stimulation withthrombin. Images were quantitated using NIH Image. Data from at least 70individually recorded cells were used to calculate the changes influorescence (y-axis). See FIG. 16A, which presents fluorescence in([Ca⁺⁺]_(i) level) increase 30 seconds after thrombin addition. Each barin FIG. 16A represents the mean ((F_(S)−F_(B)/F_(B)−1)±SEM of over 70individually recorded cells. The ** symbol indicates p<0.005. FIG. 16Bshows the kinetics of [Ca⁺⁺]_(i); fluorescence changes after cellstimulation with thrombin. Data presented are the mean((F_(S)−F_(B)/F_(B)−1)±SEM at each recording point for cells transfectedwith CDNA for the empty vector (pcDNA) or the Gq C-terminal minigenevector (pcDNA-Gαq). The arrow indicates the time thrombin was added.Each time point represents over 100 individually recorded cells.

As shown in FIG. 16, following cell activation by addition of thrombinthere was a transient increase in intracellular [Ca⁺⁺]_(i) levels.Thirty seconds after the addition of thrombin, cells transfected withpcDNA-Gαq had a calcium response that was 44% decreased as compared tocells transfected with pcDNA (FIG. 16A). pcDNA-Gαq transfected cells hada 45% decrease compared to those transfected with pcDNA when all timepoints measured after thrombin stimulation are averaged (FIG. 16B). Thisdecrease appears to be specific as cells transfected with pcDNA-Gαi orpcDNA-GαiR did not have any effect on thrombin stimulated [Ca⁺⁺]_(i)levels. Thus, cells expressing the Gαq C-terminal peptide appear to beinhibited in their ability to stimulate [Ca⁺⁺]_(i) levels followingactivation with thrombin, indicating a specific block of this downstreammediator by expression of Gαq.

pcDNA, pcDNA-GiR, pcDNA-Gi, pcDNA-Gq, or pcDNA-Gs minigene constructswere transfected into HMEC and used to assay inositol phosphate (IP)accumulation. After 24 hours, cells were reseeded onto 24-well platesand labeled with [³H]-myoinositol (2 μCi/ml). After 48 hours, cells wererinsed, and incubated with or without thrombin (10 nM) for 10 minutes.Total IP accumulation was assayed as described above using Dowex™columns to separate [³H]IP. The relative amount of [³H]IP generated wascalculated as follows: ([³H]IP (cpm)/[³H]IP (cpm)+[³H]inositol (cpm)).Each value was normalized by the basal value (no thrombin stimulation)obtained in pcDNA transfected cells. See FIG. 17. The results presentedare the normalized mean±SEM of at least three independent experimentsperformed in triplicate. The ** symbol indicates p<0.005.

Example 27 Prevention of Thrombin-Induced MAPK Activity by GPCR-bindingC-terminal Peptides

Hemagglutanin (HA)-MAPK (1×10⁵/mL was co-transfected into HMEC with thepcDNA, pcDNA-Gαi, pcDNA-Gαq or pcDNA-GαiR minigene constructs using themethods described in Example 24. After 30 hours, cells wereserum-starved for 18 hours and then treated with 10 nM thrombin for 20minutes. Cells were then lysed with RIPA buffer (50 mM Tris, pH 7.5, 150mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 10%glycerol, 10 μg/mL aprotinin and 10 μg/mL leupeptin) and HA-MAPK proteinimmunoprecipitated using 12CA5 antibody (Roche Molecular Biochemicals;Indianapolis, Ind.) (one hour, 4° C.) and Protein A sepharose beads(three hours, 4° C.). Immune complexes were washed three times in RIPAbuffer. Kinase activity in the immunoprecipitates was measured usingmaltose binding protein (MBP) substrate and a kinase assay kit (UpstateBiotechnology, Inc., Lake Placid, N.Y.). MAPK activity (nmol/min/mg) wasobtained for each, and the relative increase of MAPK activity(thrombin-mediated fold increase) was calculated as follows: (stimulatedactivity (nmol/min/mg)−basal activity (nmol/min/mg))/basal activity(nmol/min/mg). Results are presented as the mean±SEM of at least threeindependent experiments in FIG. 18. A * symbol indicates p<0.05.

Addition of 10 nmol thrombin resulted in a 3.66 fold increase in HA-MAPKactivity in cells transfected with the pcDNA control vector. Similarly,cells transfected with pcDNA-GiR had an essentially equivalent increasein thrombin mediated MAPK activity with (4.46 fold increase). However,endothelial cells transfected with a minigene construct encoding theGαi, Gαq, Gα12 or Gα13 GPCR binding peptides showed a significantdecrease in thrombin-mediated HA-MAPK activity (59%, 57%, 50% and 77%,respectively) compared to cells transfected with pcDNA.

Example 28 Reduction of Thrombin-Induced Transendothelial ElectricalResistance

Transendothelial electrical resistance (TEER) was measured by passing analternating current (50 μA; 2 pulses every minute) across monolayers ofHMEC expressing Gαq, Gαi, GαiR or no minigene construct. Basal TEER didnot change significantly with minigene transfection. Upon addition of 10nM thrombin, however, there was a decrease in the TEER of cellsexpressing the Gαq minigene compared to non-transfected cells in thepresence of 10 nM thrombin. See FIG. 19 (representative of multipleexperiments). The decrease in transendothelial electrical resistance inresponse to thrombin was significantly reduced in endothelial cellstransfected with the minigene for the carboxyl terminus of Gαq, whilethere was no effect in cells transfected with Gαi, GαiR, or emptyvector. These results suggested that Gαq is partially responsible forthe effects of thrombin on endothelial cell shape changes.

Example 29 Inhibition of Thrombin-Mediated Stress Fiber Formation

HMEC cells were transfected with pcDNA, pcDNA-Gα12 or pcDNA-Gα13minigene constructs 1 μg each/100 mm dish. As a marker for transfectedcells, the pGreenLantern-1 plasmid, containing the gene for greenfluorescent protein (GFP) was co-transfected together with minigeneconstructs. After 48 hours, cells were serum starved for 18 hours andtreated with 10 nM thrombin for 20 minutes. After exposure to thrombin,the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1%Triton X-100 and stained for F-actin with 1 mM rhodamine-phalloidin for30 minutes. Cells were extensively washed, mounted using Vectashield™antifade mounting medium (Vector Laboratories, Inc.). Cells wereobserved with an inverted microscope (Diaphot 200, Nikon, Inc.) equippedfor both differential interference contrast microscopy andepifluorescence observation using a 60x oil-immersion objective.Fluorescence and DIC images were recorded for each cell field with acooled, integrating CCD array camera (Imagepoint, Photometrix, Ltd.)connected to the microscope. See FIG. 20 for fluorescence images showinginhibition of thrombin-mediated stress fiber formation by Gα12 and Gα13peptides.

Serum-starved cells transfected with pcDNA exhibited a thin corticalF-actin rim at their margins, and contained few stress fibers (FIG. 20,panel A). Those present were inconspicious and in apparently randomorientation. For HMEC transfected with pcDNA after a 20-minute exposureto thrombin actin had reorganized into prominent stress fibers,typically arranged in a parallel pattern along the longitudinal axis ofthe cell (FIG. 20, panel B). A very different pattern was observed forcells transfected with pcDNA-Gα12 (FIG. 20, panel C) or pcDNA-Gα13 (FIG.20, panel D) minigenes after exposure to thrombin. In both pcDNA-Gα12and pcDNA-Gα13 transfected cells, thrombin stimulation did not result inthe appearance of stress fibers. In cells transfected with pcDNA-Gα13,the peripheral actin rim appears thicker and more linear, providing aclear outline of cell-cell junctions. Thus, in agreement with earlierreports, thrombin induced rapid stress fiber formation in endothelialcells. Transfection of either pcDNA-Gα12 or pcDNA-Gα13 minigenesresulted in cells that no longer showed thrombin-induced stress fiberformation. Given that stress fiber formation is dependent on the smallGTPase Rho, these results concur with other findings that Gα12 and Gα13are intimately linked to Rho signaling and demonstrates the ability ofGPCR binding peptides to specifically block this G protein pathway whenexpressed intracellularly.

Example 30 Inhibition of G Protein Activity by GPCR Binding Peptides inSingle Intact Cells

Human embryonic kidney (HEK) 293 cells, which stably express the M₂ mACR(˜400 fmol receptor/mg protein), were grown in DMEM (Gibco) supplementedwith 10% fetal bovine serum (Gibco), streptomycin/penicillin (100 Ueach; Gibco) and G418 (500 mg/L; Gibco). Cells were grown under 10% CO₂at 37° C. In all transfections for electrophysiological studies, the CD8reporter gene system was used to visualize transfected cells usingDynabeads™ coated with anti-CD8-antibodies (Dynal). The followingamounts of cDNA were used to transfect the cells: pC1-GIRK1 (rat)—1 μg;nH3-CD* (human)—1 μg; pcDNA3.1, pcDNA-Gαi, pcDNA-GαiR, pcDNA-Gαq, orpcDNA-Gαs—4 μg. Thus, typically the total amount of cDNA used fortransfecting one 10 cm disk was 7 μg. The cDNAs for GIRK1 and GIRK4 weregifts from F. Lesage and M. Lazdunski (Nice, France). A standard calciumphosphate procedure was used for transient transfection of HEK cellsaccording to the methods of Schenborn et al., Meth. Mol. Biol.130:135-145, 2000. All assays were performed 48-72 hours posttransfection.

Whole cell currents were recorded from stably M₂ mAChR-expressing HEK293 cells that had been transiently transfected with cDNA for GIRK1,GIRK4 and either pcDNA-Gαi, pcDNA-Gαs, or pcDNA-Gαq. For the measurementof inwardly rectifying K⁺ currents, whole cell currents were recordedusing an extracellular solution contained 120 mM NaCl; 20 mM KCl; 2 mMCaCl₂; 1 mM MgCl₂; and 10 mM HEPES-NaOH, pH 7.4. The solution forfilling the patch pipettes was composed of 100 mM potassium glutamate;40 mM KCl; 5 mM MgATP; 10 mM HEPES-KOH, pH 7.4; 5 mM NaCl; 2 mM EGTA; 1mM MgCl₂; and 0.01 mM GTP. Membrane currents were recorded under voltageclamp, using conventional whole cell patch techniques. See Bunemann etal., J. Physiol. 489:701-777, 1995 and Bunemann et al., J. Physiol.482:81-89, 1995, the disclosures of which are hereby incorporated byreference. To minimize variations due to different transfections orculture conditions, control experiments (transfection with pcDNA-GαiR)were done in parallel. Patch-pipettes were fabricated from borosilicateglass capillaries, (GF-150-10, Warner Instrument Corp.) using ahorizontal puller (P-95 Fleming & Poulsen). The DC resistance of thefilled pipettes ranged from 3-6 MΩ.

Membrane currents were recorded using a patch-clamp amplifier (Axopatch200, Axon Instruments). Signals are analog-filtered using a lowpassBessel filter (1-3 kHz corner frequency). Data were digitally storedusing an IBM compatible PC equipped with a hardware/software package(ISO2 by MFK, Frankfurt/Main, Germany) for voltage control, dataacquisition and data evaluation. To measure K⁺ currents in the inwarddirection, the potassium equilibrium potential was set to about −50 mVand the holding potential was −90 mV. Agonist-induced currents wereevoked by application of acetylcholine (ACh; 1 μM) using a solenoidoperated superfusion device which allowed for solution exchange within300 mseconds. Linear voltage ramps (from −120 mV to +60 mV within 500mseconds) were applied every 10 seconds. By subtracting non-agonistdependent currents, the current voltage properties of the agonistinduced currents could be resolved. To exclude experiments in whichcurrents were recorded from cells that may not have expressed thefunctional channel, only those cells that exhibited a basal non-agonistdependent Ba⁺⁺ (200 μM) sensitive inwardly rectifying current were usedfor analysis. For analysis of the data, the maximal current density(peak amplitude) of ACh-induced inwardly rectifying K⁺ currents wasmeasured at −80 mV and compared.

Superfusion of HEK 293 cells transiently transfected with GIRK1/GIRK4and either pcDNA-Gi or pcDNA-GiR DNA with 1 μM ACh revealed that cellstransfected with pcDNA-Gαi DNA have a dramatically impaired response tothe M₂ mAChR agonist. See FIG. 21, which summarizes data showing themaximum amplitude of ACh evoked currents for the different transfectionconditions (cells transfected with GIRK1/GIRK4 and pcDNA-Gi or cellstransfected with GIRK1/GIRK4 and pcDNA-GiR). The pcDNA-Gi minigenevector results in high intracellular expression of the Gαi peptide,leading to diminished ability of the receptor to signal theheterotrimenic Gαi.

The maximum current evoked by ACh was 3.7+/−1.5 pA/pF (n=14) in cellstransfected with pcDNA-Gi, compared to 24.1+/−8.8 pA/pF (n=11) in cellstransfected with pcDNA-GiR. This indicates that the Gαi minigeneconstruct completely blocked the agonist mediated M₂ mAChR GIRK1/GIRK4response while the control minigene construct (pcDNA-GiR) had no effect.Compare FIG. 21A to FIGS. 21B and 21C. Cells transfected with minigeneconstructs encoding Gα carboxyl termini for Gαq or Gαs pcDNA-Gαq orpCDNA-Gαs were not significantly different than those of cellstransfected with the control vectors. These findings confirm thespecificity of the inhibition of M₂ mAChR-activated G protein-coupledinwardly rectifying K⁺ current responses by expression of the Gαiminigene.

Example 31 Selective G Protein Signaling Inhibition in HumanMicrovascular Endothelial Cells

Different measures of G-protein signaling final actions were assayed inhuman microvascular endothelial cells (HMEC) which natively express thethrombin receptor, PAR1. The cells were seeded onto 6-well plates at1×10⁵ cells/well and transiently transfected after 24 hours withminigene constructs containing Gα carboxyl terminal peptides (pcDNA,pcDNA-Gαi, or pcDNA-GαiR; 1 μg per well) using Effectene (Qiagen)according to the manufacturer's protocol. After 24 hours, the cells werelabeled with 3 μCi/ml [³H]-adenine for 30 minutes at 37° C. Afteranother 24 hours, the cells were washed with serum-free mediumcontaining 1 mM isobutyl-methyl xanthine. To stimulate cAMPaccumulation, cells were treated with 1 μM isoproterenol for 30 minutesat 37° C. To see the inhibitory effects of thrombin on cAMPaccumulation, cells were pretreated with thrombin (50 nM) for 15 minutesprior to addition of isoproterenol. The reactions were terminated byaspiration of media followed by addition of ice-cold 5% trichloroaceticacid.

Results are provided in FIG. 22 as (cAMP/cAMP+ATP) X 1000. Threeseparate experiments were done in duplicate. The ** symbol indicatesp<0.005. Basal cAMP levels were essentially equivalent for allconditions tested. Endothelial cells stimulated with isoproterenol toactivate β-adrenergic receptors increase their cAMP levels through theGs pathway. Cells transfected with pcDNA, pcDNA-Gαi, or pcDNA-GαiRshowed little difference with 82-, 64-, and 77-fold increases inisoproterenol-mediated cAMP accumulation, respectively. When theendothelial cells were pre-incubated with thrombin prior to addition ofisoproterenol, a decrease in cAMP levels was observed due to thrombinactivation of the Gi pathway. Endothelial cells transfected with pcDNAand pre-incubated with thrombin showed a 39% decrease in cAMP level overcells stimulated with only isoproterenol. Similarly, cells transfectedwith pcDNA-GαiR and pre-incubated with thrombin showed had a 43%decrease over cells stimulated with only isoproterenol. However, cellstransfected with pcDNA-Gαi and pre-incubated with thrombin had only a0.1% decrease in cAMP levels as compared to cells stimulated with onlyisoproterenol. Thus, cells expressing the Gαi C-terminal peptide appearto be unable to inhibit adenyl cyclase following activation withthrombin, indicating that thrombin-mediated Gi signaling wasspecifically blocked by expression of the pcDNA-Gαi minigene.

Example 32 Screening Method to Identify Inverse Agonists

Urea-washed rod outer segment membrane fragments containing rhodopsinreceptor are immobilized onto microtiter wells and blocked as describedin Example 7. The receptor is light-activated. Labeled native Gαtcarboxyl terminal peptide is added to each well and allowed to shakegently for one hour at 4° C. The wells are washed to remove unboundpeptide. Crude bacterial lysates (labeled) from a Gαt carboxyl terminalpeptide prepared according to the methods described in Example 7 (200μL) are added to each well and incubated with shaking for one hour at 4°C.

The wells then are washed to remove unbound label. The supernatants orwell-bound labels are quantitated by ELISA to detect dissociation oflabeled native peptide from the receptor after incubation with librarypeptides compared to control wells incubated in the absence of librarypeptides.

Example 33 Small Molecule Library Screening Method

Small molecule libraries are screened for inhibition of GPCR-mediated Gprotein signaling as follows. PAR1 thrombin receptor prepared frominsect cells according to Example 3 are immobilized onto microtiterwells, blocked and washed. A small molecule library purchased from ChemDiv (San Diego, Calif.) are added simultaneously with MBP-peptide fusionprotein (0.1-1000 nM) in a 96- or 384-well plate and allowed to shakefor one hour at 4° C. Initial screens are performed with the smallmolecules at about 5-5000 nM. The wells are washed four times in coldPBS containing 0.05% Tween 20™ and 1 mM maltose. The amount of maltosebinding protein adhering to the wells is quantitated with anti-MBPantibodies as described in Example 17, versus control wells incubatedwithout library compounds.

Example 34 Identification of Very High Affinity ActivatedRhodopsin-Binding Gt-Based Peptides

A combinatorial peptide library based on the C-terminal sequence of Gtwas constructed by introducing all possible mutations at each position,but with an overall bias toward the Gαt sequence with a K341R change andpanned for high-affinity binding. See Martin et al., J. Biol. Chem.271:361-366, 1996; Gilchrist et al., Methods Enzymol. 315:388-404, 2000the disclosures of which are hereby incorporated by reference, andExamples 7 and 17 for methods used. Specific residues within theC-terminal sequence were highly conserved. Perhaps more interesting isnot only the selection against the native amino acid at a given position(R341, the second residue in the peptide shown below) but the apparentselection for a specific amino acid at that location (leucine). SeeTable XX. Table XXI shows amino acid sequences obtained from screeningdark-adapted bovine rhodopsin with the same combinatorial peptidelibrary based on the C-terminal sequence of Gt. As observed with Gt,specific residues within the carboxyl terminal sequence were conservedand specific residues were selected against. Notably, at identicalpositions there are extreme differences between the selection oflight-activated and dark-adapted rhodopsin (i.e., position C347)indicating that upon activation the receptor undergoes a conformationalchange unmasking new sites which the G protein can interact. TABLE XXAlignment of the Highest-Affinity Amino Acid Sequences Screened based onthe C-Terminal Sequence of Gt with Light-Activated Rhodopsin.

TABLE XXI Alignment of Amino Acid Sequences Screened based on theC-Terminal Sequence of Gt with Dark-Adapted Rhodopsin.

In all the high affinity sequences selected for binding to the lightadapted rhodopsin, position 341, which normally is a positively chargedresidue was changed to a neutral one. There is an obvious selection fora specific amino acid change from R to L. Peptides synthesized with thissingle change were assayed for high affinity binding, and the resultsare shown in Table XX. There was not a selection for a neutral aminoacid at position 341 in peptides selected for binding to dark-adaptedrhodopsin. See Table XXI. Arg is found 75% of the time. For peptidesselected for light-activated rhodopsin, Leu344, Cys347, and Gly348 werefound to be invariant, and hydrophobic residues were always located atpositions 340, 349 and 350, indicating the critical nature of theseresidues. This differs considerably from the peptides selected forbinding to dark-adapted rhodopsin, which did not show any invariantpositions. Most striking is the apparent selection against the Cys347position. Cys347 and Gly 348 both are part of a type II′ β-turn which isrequired for MII stabilization. This suggests that a site on rhodopsinwhich is required to bind the Cys 347 of Gt is unmasked only after thereceptor has received a photon of light and formed MII. See Gilchrist etal., Methods Enzymol. 15:388, 2000. Other works indicate that thecritical nature of Cys347 for binding light-activated rhodopsin is dueto its hydrophobicity. Aris et al., J. Biol. Chem. 276:2333, 2001.Replacement of Cys 347 with a hydrophobic amino acid (Cys347Met,Cys347Val and Cys347Abu)(Abu=2-aminobutyric acid) stabilizes MII to thesame extent and with similar potency as the parent peptide. The apparentselection of Lys at 347 in the dark-adapted rhodopsin peptides clearlyindicates that binding of the Gαt peptide to dark-adapted rhodopsin isvery different from light-activated rhodopsin. These results show thatthe site on rhodopsin recognized by the C-terminal tail of Gt differsdepending on whether the receptor is dark-adapted or light-activated.This implies that high affinity peptides selected for binding tolight-activated rhodopsin only bind the activated state of the receptorand not dark-adapted receptor.

Example 35 Assays for Determining Peptide, Peptide-Fusion Protein orSmall Molecule Affinities for Metarhodopsin II

For the “extra MII” assay, EDTA-washed rhodopsin (Example 5; 5 μM) isincubated in a 50 mM HEPES buffer, pH 8.2, with 100 mM NaCl, 1 mM MgCl₂,and 1 mM DTT at 5.3° C., in the absence or presence of varyingconcentrations of Gt340-350 analogs or Gt. The sample is maintained at5.4° C. using a water-jacketed and thermostated circulator cuvetteholder in an SLM Aminco DW2000 spectrophotometer at 390 and 440 nm. Aflash of light bleaching 10% of the rhodopsin is presented and after a 1min incubation, a second spectrum is measured and the difference inspectrum calculated. “Extra” MII is calculated as the difference betweenthe absorbance at 390 and 440. Dose response curves of MII stabilizationby αt340350 (λ), mutant α340-350K341L(ν), and heterotrimeric Gt (v) wereanalyzed by non-linear regression using the program GraphPad PRISM andare shown in FIG. 23.

For the MII decay assay, the absorbance spectra of EDTA-washed ROS (10μM) is measured in an SLM Aminco DW2000 spectrophotometer in 10 mMK₂PO₄, pH 6.5 containing 0.1 M KCl, 0.1 mM EDTA, 1 mM DTT, in thepresence of peptide, fusion protein expressing high affinity peptide orsmall molecule. The spectra are measured in the dark, then completelybleached in room light. The spectra of the bleached sample is measuredat intervals of 30 minutes over a 6 hour period.

Example 36 Analysis of Data From Small Molecule Library Screen

Competition ELISA assays were employed to screen a small moleculelibrary (a 10,000 compound library representative of ChemDiv's DiverseCollection of drug-like molecules) for compounds that bind activatedrhodopsin and increase/decrease the binding of MBP-8 high affinitypeptide fusion protein. MBP 8 was selected based on its mid-rangeaffinity. The screen may be repeated using an MBP which displays higheraffinity and ability to stabilize MII (i.e., MBP 18; Table XIX). Thesetypes of screens may be used with libraries of any size, therefore it ispossible to increase the size of the compound library by 10 fold orgreater and continue screening for small molecule hits in a similarmanner.

A software program that displays results of screening as a colorometricreadout with a unique color coding that represents the amount ofinhibition or stimulation of bound light-activated rhodopsin-boundpeptide fusion protein is advantageous and preferred. Two representative96-well plates in which light-activated-rhodopsin-bound MBP-8high-affinity peptide fusion proteins were assayed for competitivebinding by 80 different compounds. Experiments were done in duplicate,and the results of the two separate plates averaged.

Dose response curves of MII (FIG. 24) indicate that both PL_(—)0302R3.C4 (σ), and heterotrimeric Gt (ν) stabilize the active form ofrhodopsin. EDTA-washed rhodopsin (5 μM) was incubated in a 50 mM HEPESbuffer, pH 8.2, with 100 mM NaCl, 1 mM MgCl₂, and 1 mM DTT at 5.3° C.,and “extra” MII was measured. For compounds that enhanced MBP-8 bindingover 25% using the color coded readout, dose studies were performed togenerate EC₅₀ curves. Table XXII below provides the calculated EC₅₀ formetarhodopsin II stabilization of each compound. TABLE XXII EC₅₀ valuesfor selected small molecules on the binding of MBP-8 to MII. Smallmolecule MW Binding of MBP-8 Name (daltons) EC₅₀ (μM) PL_0568 R1.C5291.2 0.96 PL_0551 R8.C1 328.5 0.95 PL_0894 R3.C7 424.9 10.1 PL_0302R3.C4 290.27 11.9 PL_1012 R2.C1 433.5 5.12

Example 37 Very High Affinity Agonists for Rhodopsin Have No Effect onPAR-1-Stimulated Ca⁺⁺ Transients

Small molecules PL_(—)0568 R1.C5, PL_(—)0551 R8.C1, PL-0894 R3.C7,PL_(—)0302 R3.C4, and PL_(—)1012 R2.C1 were tested for their effect onthe ability of an unrelated receptor (PAR1) to activate Ca⁺⁺ signaling.Human embryonic kidney cells were cultured in a 96-well format andallowed to adhere for 2 hours. The medium was aspirated and the plateincubated at 37° C. for 30 minutes in 0.5 mL loading buffer (20 mM HEPES(pH 7.4), 130 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 0.83 mMNa₂HPO₄, 0.17 mM NaH₂PO₄, 1 mg/ml BSA, 25 mM mannose) containing 0.1%(v/v) Pluronic F127 and 10 μM Oregon Green Bapta-1 acetoxymethyl ester.The small molecules were added to the appropriate wells after 30 minutesand the cells incubated at 37° C. for another 30 minutes. The 96-wellplate was tested for calcium concentration using a Flexstation™ system.Basal conditions were established before addition of thrombin (±70 nM).Recordings were made every 5 seconds and continued for >100 secondsafter stimulation with thrombin. See FIG. 25.

Example 38 Modulation of MBP-8 Binding to Rhodopsin by Small Molecules

To test the effects of the small molecules in cells, light responseexperiments were carried out on isolated rods from the dark-adaptedretina of a salamander. Single rods were isolated by shredding a smallpiece of retina. Photoreceptors were mechanically isolated from thedark-adapted retinas and placed in a gravity-fed superfusion chamber onthe stage of an inverted microscope. Membrane currents were recordedwith a suction electrode as described by Baylor et al., (Baylor et al.,J. Physiol. (Lond.). 288:589-611, 1979; Baylor et al. J. Physiol.(Lond.). 288:589-611, 1979) in Ringer solution containing 120 mM NaCl,2.0 mM KCl, 2 mM NaHCO₃, 1.6 mM MgCl₂, 1.0 mM CaCl₂, 10 mM glucose, and3 mM HEPES, pH 7.6, as described by Rieke and Baylor, Biophys. J.71:2553-2572, 1996. Membrane current collected by the suction electrodewas amplified, low-pass filtered at 20 Hz (3 dB point; 8-pole Bessellow-pass), digitized at 100 Hz and stored on a computer for subsequentanalysis. Light responses were elicited by 10-msecond flashes of 50-500nm light. The flash strength was controlled with calibrated neutraldensity filters. The cell was positioned in the suction electrode tocollect as much dark current as possible. Solution changes (by whichaddition of the small molecule was effected) were achieved with a seriesof electronically controlled pinch valves (Biochem Valves, Boonton,N.J.) the outlets of which were connected to a common perfusion pipeabout 100 μm in diameter. Solution changes with this system werecompleted in 200-300 mseconds. Solutions were driven by positivepressure through a pair of glass pipes with openings about 50 μm indiameter. The pipes were mounted on a piezoelectric translation stage(Burleigh Instruments, Fishers, N.Y.). Solution changes at the cut endof the outer segment were completed in less than 10 mseconds with thissystem.

Light stimuli were delivered from a dual beam optical bench.Monochromatic lights were obtained by passing the light from atungsten-halogen bulb through interference filters with 10 nm nominalbandwidths. Wavelength (520 nm for rods and 440 nm or 620 nm for cones)and intensity of the stimulating light were set with calibrated narrowband interference and neutral density filters, respectively. SalamanderL, S, and ultraviolet-sensitive cones have peak sensitivities at 600 nm,430 nm, and 360 nm (Makino and Dodd, J. Gen. Physiol. 108:27-34, 1996)and are readily identified by the relative amplitudes of their responsesto 620 nm, 440 nm, and 380 nm lights. After identification, S cones werestimulated with 440 nm light and L cones were stimulated with 620 nm.Ultraviolet-sensitive cones were not studied. Light intensities werecontrolled with a set of calibrated neutral density filters, and lightflashes were produced by an electronically controlled shutter in thelight path.

The results are presented in FIGS. 26 and 27, which show the lightresponse of an isolated rod from the dark-adapted retina of a salamanderin the presence or absence of 5 μM compound PL_(—)0302R3C4,respectively. Panel A of each figure shows the membrane current(response) plotted against time for the light responses as a result ofincreasing light flashes. In panel B, the peak responses have beennormalized so that the current at the highest light flash is 1.0. Thecircles correspond to the peak response for each light flash. In FIG.27, panel B, the bissecting lines indicate that in the presence ofPL_(—)0302R3C4 a lower intensity is required to get the same change ofcurrent. The results indicate that compound PL_(—)0302R3C4 increases thepeak response (as measured by a change in current) 20%-50%, depending onthe intensity of the light flash and thus the amount of rhodopsinreceptors activated. The results are representation of these separateexperiments. The results suggest taht the compound PL_(—)302R3C4 canserve as an allosteric agonist and increase the signaling activity ofthe receptor in cells.

Small molecules also were tested for their ability to enhance thebinding of MBP-8. PELM6 (the MBP control) and MBP-8 were plated on 96well plates that contained EDTA-washed rhodopsin. A small moleculecompound library was added, and the amount of pELM6 or MBP-8 thatremained bound was measured. Standard methods were used.

FIGS. 28-32 show MBP-8 binding curves for the depicted small molecules'ability to enhance binding of the high affinity peptide fusion protein,MBP-8 to EDTA-washed rhodopsin.

1. A two-screen method of detecting high-affinity G protein coupledreceptor (GPCR) G protein interaction site binding compounds, whichcomprises: (a) providing a peptide library the members of which arebased on the primary sequence of a native G protein Gα subunit carboxylterminal peptide sequence that binds to said GPCR on a G proteininteraction site of said GPCR, wherein said G protein Gα subunitcarboxyl terminal peptide sequence is selected from the group consistingof SEQ ID NOs: 2, 13, 15, 17, 21, 25, 26, 27, 30, 34, 38, 40, and 45-85;(b) screening said peptide library for in vitro high affinity binding tosaid G protein interaction site of said GPCR to identify library membersthat bind to said G protein interaction site with higher affinity thanthat of said native G protein Gα subunit carboxyl terminal peptidesequence; (c) selecting a member of said peptide library having bindingto said GPCR of higher affinity than that of said native G protein Gαsubunit carboxyl terminal peptide sequence (d) providing a library ofcandidate compounds to screen for binding to said G protein interactionsite of said GPCR; (e) screening said library of candidate compounds invitro for binding to said GPCR in competition with a member of saidpeptide library selected in step (c) to detect candidate compoundshaving high-affinity binding to said G protein interaction site of saidGPCR.
 2. A two-screen method of detecting high-affinity GPCR G proteininteraction site binding compounds, which comprises: (a) providing apeptide library the members of which are based on the primary sequenceof a native G protein Gα subunit carboxyl terminal peptide sequence thatbinds to said GPCR on a G protein interaction site of said GPCR; (b)screening said peptide library for in vitro high affinity binding tosaid G protein interaction site of said GPCR to identify library membersthat bind to said G protein interaction site with higher affinity thanthat of said native G protein Gα subunit carboxyl terminal peptidesequence; (c) selecting a member of said peptide library having bindingto said GPCR of higher affinity than that of said native G protein Gαsubunit carboxyl terminal peptide sequence; (d) providing a library ofcandidate compounds to screen for binding to said G protein interactionsite of said GPCR; (e) screening said library of candidate compounds invitro for binding to said GPCR in competition with a member of saidpeptide library selected in step (c) to detect candidate compoundshaving high-affinity binding to said G protein interaction site of saidGPCR.
 3. A method of claim 1, wherein said screening of step (b) isperformed by testing for binding to an intact G protein coupledreceptor.
 4. A method of claim 1, wherein said screening of step (b) isperformed by testing for binding to an intracellular fragment of a GPCR.5. A method of claim 1, wherein said screening of step (b) comprises acompetitive binding assay.
 6. A method of claim 5, wherein saidcompetitive binding assay is characterized by co-incubation of membersof said peptide library with said native Gα subunit carboxyl terminalpeptide sequence.
 7. A method of claim 1, wherein binding to said GPCRis determined by measuring a signal generated from interaction of anactivating ligand with said GPCR.
 8. A method of claim 1, wherein saidlibrary of variant peptides is a combinatorial peptide library.
 9. Amethod of claim 8, wherein said library of variant peptides is aprotein-peptide fusion protein library.
 10. A method of claim 9, whereinsaid protein-peptide fusion protein library is a maltose bindingprotein-peptide fusion protein library.
 11. A method of claim 1, whereinsaid library of variant peptides is a peptide display library.
 12. Amethod of claim 2, wherein said native G protein Gα subunit carboxylterminal peptide sequence that binds to said GPCR on a G proteininteraction site of said GPCR is from about 7 to about 70 amino acidslong.
 13. A method of claim 2, wherein said native G protein Gα subunitcarboxyl terminal peptide sequence that binds to said GPCR on a Gprotein interaction site of said GPCR is from about 7 to about 55 aminoacids long.
 14. A method of claim 2, wherein said native G protein Gαsubunit carboxyl terminal peptide sequence that binds to said GPCR on aG protein interaction site of said GPCR is from about 8 to about 50amino acids long.
 15. A method of claim 2, wherein said native G proteinGα subunit carboxyl terminal peptide sequence that binds to said GPCR ona G protein interaction site of said GPCR is from about 9 to about 23amino acids long.
 16. A method of claim 2, wherein said native G proteinGα subunit carboxyl terminal peptide sequence that binds to said GPCR ona G protein interaction site of said GPCR is about 11 amino acids long.17. A method of claim 16, wherein said G protein Gα subunit carboxylterminal peptide sequence is selected from the group consisting of SEQID NOs: 2, 13, 15, 17, 21, 25, 26, 27, 30, 34, 38, 40, and 45-85.